Advertisement

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*

Open AccessPublished:May 24, 2013DOI:https://doi.org/10.1074/jbc.M112.448225
      Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.
      Background: cytb5 modulates catalysis performed by cytsP450, in vivo and in vitro.
      Results: The structure of full-length cytb5 was solved by NMR, and the cytP450-binding site on cytb5 was identified by mutagenesis and NMR.
      Conclusion: A model of the cytb5-cytP450 complex is presented. Addition of a substrate strengthens the cytb5-cytP450 interaction.
      Significance: The cytb5-cytP450 complex structure will help unravel the mechanism by which cytb5 regulates catalysis by cytP450.

      Introduction

      Cytochromes P450 (cytsP450)
      The abbreviations used are: cytsP450
      cytochromes P450
      1-CPI
      1-(4-chlorophenyl) imidazole
      two-dimensional HSQC
      two-dimensional heteronuclear single quantum coherence
      BHT
      3,5-di-tert-butyl-4-hydroxytoluene
      cytb5
      cytochrome b5
      cytP450
      cytochrome P450
      CPR
      cytochrome P450 reductase
      DHPC
      1,2-dihexanoyl-sn-glycero-3-phosphocholine
      DLPC
      1,2-dilauroyl-sn-glycero-3-phosphocholine
      DMPC
      1,2-dimyristoyl-sn-glycero-3-phosphocholine
      DPC
      dodecylphosphocholine
      SLF
      separated local field
      TROSY
      transverse relaxation optimized spectroscopy
      PDB
      Protein Data Bank
      r.m.s.d.
      root mean square deviation.
      are a ubiquitous superfamily of mixed-function oxygenases, which are found in all kingdoms of life but are especially abundant in eukaryotes. Humans possess 57 different membrane-bound cytsP450 (
      • Guengerich F.P.
      • Wu Z.L.
      • Bartleson C.J.
      Function of human cytochrome P450s: characterization of the orphans.
      ). They are found in all tissues of the body and are responsible for influencing a dazzling array of biochemical and physiological processes, including embryonic development, blood coagulation, and the metabolism of carcinogens, environmental toxins, over 50% of drugs in use, vitamin D, and other exogenous and endogenous compounds (
      • Shen A.L.
      • O'Leary K.A.
      • Kasper C.B.
      Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome p450 oxidoreductase.
      ,
      • Nebert D.W.
      • Russell D.W.
      Clinical importance of the cytochromes P450.
      ). Selected human cytsP450 (cytP45017A1 and cytP45019A1) are targets for the treatment of prostate and breast cancer, respectively (
      • O'Donnell A.
      • Judson I.
      • Dowsett M.
      • Raynaud F.
      • Dearnaley D.
      • Mason M.
      • Harland S.
      • Robbins A.
      • Halbert G.
      • Nutley B.
      • Jarman M.
      Hormonal impact of the 17α-hydroxylase/C(17,20)-lyase inhibitor abiraterone acetate (CB7630) in patients with prostate cancer.
      ,
      • Orlando L.
      • Schiavone P.
      • Fedele P.
      • Calvani N.
      • Nacci A.
      • Rizzo P.
      • Marino A.
      • D'Amico M.
      • Sponziello F.
      • Mazzoni E.
      • Cinefra M.
      • Fazio N.
      • Maiello E.
      • Silvestris N.
      • Colucci G.
      • Cinieri S.
      Molecularly targeted endocrine therapies for breast cancer.
      ).
      CytP450 catalyzes the insertion of one atom of “activated” molecular oxygen into the substrate, using two electrons from NAD(P)H and two protons from water. Electrons destined for cytP450 are first delivered to its redox partners, cytP450-reductase (CPR) and cytb5, which then transfer the electrons to cytP450 (
      • Im S.C.
      • Waskell L.
      The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase, and cytochrome b5.
      ). CPR is capable of transferring both electrons to cytP450; however, cytb5 is capable of donating only the second electron due to its high redox potential as compared with ferric cytP450 (
      • Zhang H.
      • Hamdane D.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4.
      ,
      • Gruenke L.D.
      • Konopka K.
      • Cadieu M.
      • Waskell L.
      The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.
      ,
      • Finn R.D.
      • McLaughlin L.A.
      • Ronseaux S.
      • Rosewell I.
      • Houston J.B.
      • Henderson C.J.
      • Wolf C.R.
      Defining the in vivo role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.
      ,
      • Guengerich F.P.
      Cytochrome P450s and other enzymes in drug metabolism and toxicity.
      ). cytb5 plays a key role in the oxidation of a variety of exogenous and endogenous compounds, including drugs, fatty acids, cholesterol, and sex hormones. The influence of cytb5 on cytP450 activity has been shown to depend on the cytP450 isozyme and the substrate involved. Remarkably, cytb5 enhances some catalytic reactions of cytP450 but does not affect or even inhibit others (
      • Im S.C.
      • Waskell L.
      The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase, and cytochrome b5.
      ,
      • Gruenke L.D.
      • Konopka K.
      • Cadieu M.
      • Waskell L.
      The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.
      ,
      • Finn R.D.
      • McLaughlin L.A.
      • Ronseaux S.
      • Rosewell I.
      • Houston J.B.
      • Henderson C.J.
      • Wolf C.R.
      Defining the in vivo role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.
      ,
      • Guengerich F.P.
      Cytochrome P450s and other enzymes in drug metabolism and toxicity.
      ,
      • Shimada T.
      • Mernaugh R.L.
      • Guengerich F.P.
      Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5 enzymes.
      ,
      • Canova-Davis E.
      • Chiang J.Y.
      • Waskell L.
      Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.
      ,
      • Morgan E.T.
      • Coon M.J.
      Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5.
      ). At low concentrations, cytb5 may enhance the rate of catalysis by up to 100-fold, whereas at high concentrations it inhibits catalysis by competing with CPR for a binding site on cytP450, thereby preventing the transfer of the first electron and the reduction of ferric cytP450 to the ferrous form (
      • Im S.C.
      • Waskell L.
      The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase, and cytochrome b5.
      ,
      • Zhang H.
      • Hamdane D.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4.
      ,
      • Zhang H.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.
      ). cytb5 and CPR are both negatively charged proteins, which are known to have overlapping binding sites on cytP450 (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). When the stimulatory and inhibitory effects of cytb5 are equal, cytb5 will appear to have no effect on the catalytic activity of cytP450.
      To obtain an in-depth understanding of the molecular basis of the effects of cytb5 on cytP450 activity, it is necessary to determine the structure of the complex between the full-length forms of cytb5 and cytP450. Although all reported x-ray and NMR structural data pertain to cytosolic heme binding domains of truncated, microsomal cytP450 and cytb5, in which their membrane anchors have been removed (
      • Halpert J.R.
      Structure and function of cytochromes P450 2B: from mechanism-based inactivators to x-ray crystal structures and back.
      ,
      • Scott E.E.
      • He Y.A.
      • Wester M.R.
      • White M.A.
      • Chin C.C.
      • Halpert J.R.
      • Johnson E.F.
      • Stout C.D.
      An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution.
      ,
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ,
      • Nunez M.
      • Guittet E.
      • Pompon D.
      • van Heijenoort C.
      • Truan G.
      NMR structure note: oxidized microsomal human cytochrome b5.
      ), neither structures nor dynamics of the full-length protein (containing the transmembrane domain) are currently available. Additionally, although the interaction of cytb5 with various membranes has been previously studied (
      • Başaran N.
      • Doebler R.W.
      • Goldston H.
      • Holloway P.W.
      Effect of lipid unsaturation on the binding of native and a mutant form of cytochrome b5 to membranes.
      ,
      • Greenhut S.F.
      • Taylor K.M.
      • Roseman M.A.
      Tight insertion of cytochrome b5 into large unilamellar vesicles.
      ,
      • Chester D.W.
      • Skita V.
      • Young H.S.
      • Mavromoustakos T.
      • Strittmatter P.
      Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.
      ), the structure of membrane-bound cytb5 is lacking. However, only the full-length membrane-binding form of microsomal cytb5 influences the enzymatic activity of cytP450 (
      • Clarke T.A.
      • Im S.C.
      • Bidwai A.
      • Waskell L.
      The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
      ,
      • Chudaev M.V.
      • Gilep A.A.
      • Usanov S.A.
      Site-directed mutagenesis of cytochrome b5 for studies of its interaction with cytochrome P450.
      ); truncated cytb5 is only capable of electron transfer to water-soluble oxidative enzymes (e.g. cytochrome c and metmyoglobin) (
      • Vergéres G.
      • Waskell L.
      Cytochrome b5, its functions, structure, and membrane topology.
      ).
      Despite recent advances in NMR methodology and isotopic labeling schemes, the structure determination of large membrane-bound protein-protein (∼70 kDa) complexes remains a monumental task. The large size of the membrane-bound complex presents considerable challenges in terms of sample stability, spectral sensitivity, and resolution. In this study, we present the first full-length tertiary structure of rabbit ferric
      Unless otherwise stated, all the NMR data were collected on the oxidized (ferric; Fe(III)) form of full-length cytP450 and cytb5.
      cytb5 solved in a membrane mimetic using a combination of high resolution solution and solid-state NMR spectroscopy. Subsequently, experimentally derived restraints from NMR, site-directed mutagenesis, and double mutant cycle data, obtained on the full-length proteins, were then used to generate the structure of the complex between ferric microsomal rabbit cytP4502B4 (56 kDa) and cytb5 (16 kDa), using a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking) (
      • de Vries S.J.
      • van Dijk A.D.
      • Krzeminski M.
      • van Dijk M.
      • Thureau A.
      • Hsu V.
      • Wassenaar T.
      • Bonvin A.M.
      HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
      ). The extensive structural knowledge of the cytb5-cytP450 complex interface provided here will prove to be essential in unraveling the molecular mechanism by which cytb5 regulates the rate of catalysis of cytP450 (
      • Zhang H.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.
      ).

      RESULTS

      Rabbit cytb5 is composed of a large cytosolic heme domain and a C-terminal transmembrane domain, connected by a 15-residue linker. To identify cytb5 residues involved in complex formation with cytP4502B4, we first solved the three-dimensional structure of ferric cytb5 in DPC micelles, using a combination of high resolution solution and solid-state NMR experiments (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1NMR structure of rabbit microsomal cytb5. A, NMR structure of full-length cytb5 obtained from a combined solution and solid-state NMR approach. The soluble heme domain structure (residues 1–104) of full-length cytb5 was solved in DPC micelles by solution NMR, with a backbone r.m.s.d. of 0.32 ± 0.10 Å. The transmembrane domain structure (residues 106–126) of full-length cytb5 was determined in aligned DMPC/DHPC bicelles using solid-state NMR spectroscopy. B, 1H-15N TROSY-HSQC spectrum of uniformly, 13C-, 2H-, and 15N-labeled cytb5 in micelles exhibiting well resolved peaks. C, two-dimensional HIMSELF spectrum of uniformly 15N-labeled cytb5 reconstituted in aligned DMPC/DHPC bicelles. The blue ring presents the best fit for the helical wheel pattern of resonances from the α-helical transmembrane domain of cytb5.

      Three-dimensional Structure Determination of Full-length Mammalian cytb5

      Isotopically labeled full-length wild-type ferric microsomal rabbit cytb5 was reconstituted in detergent (DPC) micelles. A number of standard TROSY-based multidimensional solution NMR experiments, in combination with isotopic labeling schemes, including perdeuteration, were employed to assist in resonance assignment and structure determination of cytb5. Fig. 2 presents a 1H-15N TROSY-HSQC spectrum of U-13C-, U-2H-, and U-15N-labeled full-length wild-type cytb5 incorporated in DPC micelles at 25 °C. The spectrum exhibits well resolved and dispersed NH correlations from cytb5 residues, suggesting that the protein is well folded and monodispersed in DPC micelles.
      Figure thumbnail gr2
      FIGURE 2High resolution solution NMR spectrum of cytb5. A, 1H-15N TROSY-HSQC spectrum of uniformly 13C-, 2H-, and 15N-labeled full-length mammalian cytb5 in DPC micelles. The backbone resonance peaks are labeled with the residue-specific assignment of cytb5. Unlabeled peaks include side chain resonances (Asn, Gln) and the lower populated cytb5 isomer. Tryptophan indole protons between 10 and 11 ppm were not assigned due to broadening. B, expansion of the crowded region of the 1H-15N TROSY-HSQC spectrum.
      Using standard three-dimensional solution NMR experiments, NMR resonance assignment was achieved for 88.5% of the backbone and side chain atoms of residues from the soluble domain of full-length cytb5 (Table 2). The chemical shift assignments were deposited in the Biological Magnetic Resonance Bank (code 18919). An inspection of the 1H-15N TROSY-HSQC spectrum of cytb5 revealed two or more NMR resonances for many of the residues. These two sets of NMR resonances originate from the two isomers (major and minor) of cytb5 that differ by a 180° rotation of the heme plane about the axis that cuts through the meso-carbon atoms α and γ (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ,
      • Zhang Q.
      • Cao C.
      • Wang Z.Q.
      • Wang Y.H.
      • Wu H.
      • Huang Z.X.
      The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.
      ). The ratio of the populations of the two isomers can be calculated by determining the peak intensity ratio (here in the 1H-15N TROSY-HSQC spectrum of cytb5) for identical residues in the two isomeric forms. The major/minor isomer ratio in our study for full-length rabbit cytb5 was determined to be about 6.6:1 which is similar to 5:1 ratio previously obtained for truncated rabbit cytb5 (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ) and nearly identical to the isomer ratio of 6.5:1 for truncated bovine cytb5 (
      • Zhang Q.
      • Cao C.
      • Wang Z.Q.
      • Wang Y.H.
      • Wu H.
      • Huang Z.X.
      The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.
      ). The ratio depends on the cytb5 species and has been reported to be as high as 1.5:1 for rat cytb5 (
      • Lee K.B.
      • La Mar G.N.
      • Kehres L.A.
      • Fujinari E.M.
      • Smith K.M.
      • Pochapsky T.C.
      • Sligar S.G.
      1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.
      ), despite the sequence similarity between rat and rabbit cytb5 (supplemental Fig. S1A). Although backbone assignments were done for the resonance peaks of both the major and minor isomers, all structure analyses were performed for the major isomer of ferric cytb5.
      TABLE 2Protein NMR resonance assignment for the backbone and side chain atoms of cytb5
      Cytb5 unambiguous backbone assignments for non-proline residues are as follows: 7–22, 24–32, 34–90, 92, 93, 96–99, and 101–104 (heme domain) and 106, 127, 133, and 134 (transmembrane domain)
      Cytb5-ambiguous backbone assignments as follows: 124, 126, and 128
      Residues with no assignments (non-proline residues) as follows: 1–6, 23, 33, 91, 94, 100, 105, 107–123, 125, and 129–132
      Proline residues as follows: 45, 86, 95, and 116
      NMR resonance assignments of the major isomer of cytb5 in DPC micelles revealed that the observed 1H-15N TROSY-HSQC spectrum is dominated by resonances from the soluble heme-containing domain and the flexible linker of cytb5 (Fig. 2). Residues from the transmembrane domain of cytb5 could not be identified in the 1H-15N TROSY-HSQC spectrum (Table 2). To identify the origin of the lack of transmembrane domain resonances in solution NMR, and to determine the structure and topology of the transmembrane domain of cytb5, solid-state NMR was performed on full-length cytb5 incorporated in aligned DMPC/DHPC bicelles (explained below). It is important to note here that the full-length form of cytb5 incorporated in a membrane mimetic (DPC micelles or lipid bicelles) was used for all solution and solid-state NMR measurements.

      Three-dimensional Tertiary Structure Calculation of the Heme Domain of Full-length cytb5

      A high resolution structure of the soluble domain of the major isomer of full-length ferric cytb5 (∼16 kDa) in DPC micelles was calculated using a total of 1787 NOE restraints (Table 1). Distances derived from NOE restraints, in conjunction with 39 hydrogen bonds and 112 (φ and ψ) dihedral angles, were included for structural determination into CYANA 2.1 (
      • Güntert P.
      Automated NMR structure calculation with CYANA.
      ). Twenty minimum energy conformers (Fig. 3A) of cytb5 with backbone and heavy atom (residues Lys-7 to Arg-89) r.m.s.d. values of 0.32 ± 0.10 and 0.82 ± 0.10 Å, respectively, were selected from 100 structures calculated in 10,000 annealing steps. Distance restraints used for the structural calculation and Ramachandran statistics can be found in Table 1. HADDOCK 2.1 was used to dock heme B into the 20 NMR-derived low energy structures of cytb5 (see “Experimental Procedures”) (
      • de Vries S.J.
      • van Dijk A.D.
      • Krzeminski M.
      • van Dijk M.
      • Thureau A.
      • Hsu V.
      • Wassenaar T.
      • Bonvin A.M.
      HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
      ,
      • Dominguez C.
      • Boelens R.
      • Bonvin A.M.
      HADDOCK: a protein-protein docking approach based on biochemical or biophysical information.
      ). Fifty low energy structures were obtained with no restraint violations. The 20 lowest energy HADDOCK-generated structures of cytb5 (Fig. 3) were deposited in the Protein Data Bank (code 2M33). This solution NMR structure of cytb5 was used in all subsequent analyses.
      Figure thumbnail gr3
      FIGURE 3Solution NMR structure of the cytosolic domain of full-length cytb5 in DPC micelles. A, overlay of the 20 lowest energy structures of cytb5 generated from CYANA2.1 based on the NMR restraints in . The backbone (Lys-7 to Arg-89) r.m.s.d. was 0.32 Å. B, two different views of the overlay of the 20 lowest energy structures of the heme domain of cytb5 generated from HADDOCK after docking heme B. His-44 and His-68, which coordinate the heme, are represented as green sticks. C, three different views of the structure of the heme domain of cytb5 obtained from solution NMR, with the heme B molecule orientation obtained from HADDOCK. Left, middle, and right orientations show the proximal, bottom (lower edge of the cleft), and side view of cytb5, respectively.
      The structure of cytb5 contains five α-helices, five β-strands, and one 310 helix (Fig. 3C and supplemental Fig. S1). The first β-sheet is observed for residues Lys-10 to Tyr-12 with an α-helix (α1, Leu-14 to Hi-s20) following shortly after. The heme-binding portion of cytb5 consists of two helices in the lower cleft, labeled as α4 (Thr-60 to Val-66) and α5 (Thr-70 to Phe-79), two helices in the upper cleft, labeled as α2 (Lys-39 to Glu-43) and α3 (Glu-49 to Gln-54) (Fig. 3C), and three β-strands at the bottom of the heme pocket, labeled as β3 (Lys-33 to Asp-36), β2 (Trp-27 to Leu-30), and β4 (Gly-56 to Asp-58). At the end of the structured soluble domain lies a β-strand (β5, Gly-82 to Leu-84) and a 310 helix (Pro-86 to Arg-89). The overall structure of the heme domain of full-length ferric cytb5 is found to be similar to the previously determined NMR structure of the heme domain of truncated ferric cytb5 from rabbit (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ).

      Flexible Linker Domain of cytb5 Lacks a Defined Secondary Structure

      The linker region (Ser-90–Asp-104), which connects the cytosolic heme domain of cytb5 to the transmembrane anchor, was previously characterized as random coil for various truncated forms of microsomal cytb5 that lacked a transmembrane domain (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ,
      • Nunez M.
      • Guittet E.
      • Pompon D.
      • van Heijenoort C.
      • Truan G.
      NMR structure note: oxidized microsomal human cytochrome b5.
      ); in these proteins, the C terminus had been truncated either within the linker region or beyond it. Neither intra- nor inter-residue NOEs were observed for most of the linker residues (Ser-90 to Asp-104) due to the rapid solvent exchange at those amide positions. Therefore, we show here that for full-length cytb5, incorporated in DPC micelles, the linker region is unstructured, lacking any distinct secondary structural features. A cytb5 linker region of at least 6–8 amino acids has been shown to be necessary to enable formation of a functional complex between cytb5 and its full-length redox partner cytP450 (
      • Clarke T.A.
      • Im S.C.
      • Bidwai A.
      • Waskell L.
      The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
      ). Unlike full-length cytb5, a cytb5 mutant lacking eight amino acids in the linker domain was neither able to insert efficiently into a lipid membrane (
      • Nguyen K.T.
      • Soong R.
      • Lm S.C.
      • Waskell L.
      • Ramamoorthy A.
      • Chen Z.
      Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy.
      ) nor able to form a fully functional complex with cytP450 (
      • Clarke T.A.
      • Im S.C.
      • Bidwai A.
      • Waskell L.
      The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
      ). The extended form of the cytb5 linker region, presented here, should provide the flexibility and orientational freedom necessary for efficient complex formation with its redox partners.

      Structure of the Soluble Domain of cytb5 Is Unaffected by Its Membrane Environment

      The 1H-15N TROSY-HSQC spectrum was also obtained for full-length cytb5 in 10% (w/v) DMPC/DHPC isotropic bicelles with a q ratio of 0.25. The majority of the spectrum was nearly identical to the one obtained in DPC micelles (data not shown). The largest chemical shift changes were observed for the tryptophan indole side chain (NH) resonances of Trp-109, Trp-110, and Trp-113 residues. These tryptophan residues are predicted to be at the edge of the transmembrane domain and hence should be most affected by the change in the membrane environment when going from micelles to lipid bicelles.

      Establishing the Topology and Structure of the Transmembrane Domain of Full-length cytb5 in Bicelles

      As mentioned above, the resonances for residues in the transmembrane domain of cytb5, reconstituted in either isotropic bicelles (DMPC/DHPC) or DPC micelles, were not identified in the 1H-15N TROSY-HSQC solution NMR spectra. Although sequential NOE assignments could not be carried out for the residues in the transmembrane domain, ambiguous NOE assignments, without secondary structural information, were possible for the Hα and side chain protons of residues Asn-121 to Asp-134 in solution NMR. A 1H-15N-HMQC spectrum recorded under magic angle spinning (2.5 kHz) on a selectively [15N]alanine-labeled sample of cytb5 incorporated in DPC micelles displayed broad resonances for the backbone amide-NHs of the four alanines present in the transmembrane domain of cytb5, along with narrow resonances for the alanines in the soluble domain (Fig. 4). These data suggest that the restricted motion of the transmembrane domain of cytb5 incorporated in a DPC micelle, or isotropic bicelles, causes significant broadening of the transmembrane domain resonances due to fast spin-spin relaxation. To obtain the structure of the transmembrane domain of cytb5, we employed an alternative technique of static solid-state NMR spectroscopy on uniformly 15N-labeled full-length cytb5 incorporated in bicelles (
      • Sanders C.R.
      • Hare B.J.
      • Howard K.P.
      • Prestegard J.H.
      Magnetically oriented phospholipid micelles as a tool for the study of membrane-associated molecules.
      ,
      • Dürr U.H.
      • Gildenberg M.
      • Ramamoorthy A.
      The magic of bicelles lights up membrane protein structure.
      ) composed of DMPC and DHPC lipids in a 3.5:1 molar ratio, which were magnetically aligned in the external magnetic field.
      Figure thumbnail gr4
      FIGURE 4Transmembrane domain of cytb5 is visible under magic angle spinning NMR. A, 1H-15N-HMQC spectrum recorded on a selectively [15N]alanine-labeled sample of cytb5 incorporated in DPC micelles. This spectrum was obtained from a 600 MHz Varian solid-state NMR spectrometer under a 2.5 kHz spinning speed of the sample at 37 °C, using a double-resonance magic angle spinning nanoprobe (Agilent/Varian). B, representation of cytb5 highlighting all the alanines (red sphere) in the protein.
      A two-dimensional SLF NMR experiment using the HIMSELF (
      • Dürr U.H.
      • Yamamoto K.
      • Im S.C.
      • Waskell L.
      • Ramamoorthy A.
      Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5.
      ) pulse sequence, which is based on the PIWIMz pulse scheme, was performed on magnetically aligned bicelles containing cytb5 (Fig. 1C). The resultant two-dimensional SLF spectrum correlates 15N chemical shifts with 1H-15N dipolar couplings. The two-dimensional spectrum in Fig. 1C exhibits a distinct circular PISA-wheel pattern of resonances between 60 and 100 ppm, which is indicative of an α-helical conformation and was assigned to the transmembrane anchor of cytb5 based on our previous work (
      • Dürr U.H.
      • Yamamoto K.
      • Im S.C.
      • Waskell L.
      • Ramamoorthy A.
      Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5.
      ,
      • Soong R.
      • Smith P.E.
      • Xu J.
      • Yamamoto K.
      • Im S.C.
      • Waskell L.
      • Ramamoorthy A.
      Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information.
      ). This is in agreement with previous circular dichroism (
      • Dailey H.A.
      • Strittmatter P.
      Structural and functional properties of membrane binding segment of cytochrome b5.
      ) and Fourier transform infrared (
      • Holloway P.W.
      • Buchheit C.
      Topography of the membrane-binding domain of cytochrome-B5 in lipids by Fourier-transform infrared-spectroscopy.
      ) experiments, which indicated that the transmembrane domain is at least 50% helical.
      The observed PISA wheel was empirically fitted (
      • Denny J.K.
      • Wang J.
      • Cross T.A.
      • Quine J.R.
      PISEMA powder patterns and PISA wheels.
      ) to determine the average tilt angle of the transmembrane α-helix with respect to the bilayer normal. The resonance pattern was consistent with an average tilt of 15 ± 3°, in agreement with our previously published work (
      • Dürr U.H.
      • Yamamoto K.
      • Im S.C.
      • Waskell L.
      • Ramamoorthy A.
      Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5.
      ). The value of the helix's order parameter was estimated to be 0.86. Additionally, a “structure fitting algorithm” (
      • Nevzorov A.A.
      • Opella S.J.
      Structural fitting of PISEMA spectra of aligned proteins.
      ) was used in combination with the solid-state SLF NMR data to determine the backbone structure of the transmembrane anchor as a whole, as presented in Fig. 1A. Interestingly, the transmembrane domain of cytb5 is conserved among vertebrates (sequence similarity of 78–96% (
      • Clarke T.A.
      • Im S.C.
      • Bidwai A.
      • Waskell L.
      The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
      )) and is essential for complex formation with redox partners (
      • Vergères G.
      • Waskell L.
      Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.
      ); both suggest that this α-helical domain plays an important role in the function of cytb5 and its interactions with redox partners.

      cytP4502B4-binding Epitope on cytb5 by NMR

      Perturbations in the amide-NH chemical shifts (Fig. 5) and peak heights (Fig. 6) of 15N-labeled full-length ferric cytb5 upon complex formation with unlabeled full-length ferric cytP4502B4 in isotropic bicelles composed of DMPC and DHPC lipids (DMPC/DHPC = q ratio of 0.25) were measured to identify the binding cytb5 epitope for cytP450. The addition of cytP450 to cytb5 in an equimolar ratio had two effects as follows: (a) it caused an overall reduction of the cytb5 amide signal intensities, indicating complex formation between cytP450 and cytb5, which increases the overall correlation time of cytb5 (Fig. 6A, yellow histogram), and (b) it caused modest chemical shift perturbations for cytb5 backbone amide resonances.
      Figure thumbnail gr5
      FIGURE 5Chemical shift perturbation analysis. A histogram presenting the experimentally measured changes in chemical shift values for residues in cytb5 upon complex formation with cytP450. The change in the chemical shift was calculated using the standard formula Δδ = √{(ωHNΔ1HN)2 + (ωNΔ15N)2}, where ωH = 1, ωN = 0.154, and Δδ represents the average (NH) chemical shift perturbation (
      • Popovych N.
      • Sun S.
      • Ebright R.H.
      • Kalodimos C.G.
      Dynamically driven protein allostery.
      ). The chemical shift perturbations are represented as a continuous color map on the NMR structure of cytb5. Resonances for His-32, Gly-46, His-68, and Ser-69 (represented in magenta) disappear upon complex formation.
      Figure thumbnail gr6
      FIGURE 6Mapping the effect of cytP450 binding to cytb5 measured from NMR. A, histogram representing the differential line broadening NMR data for the cytb5-cytP450 complex. The amide peak intensities for free cytb5 are presented in red. Yellow presents the intensities for cytb5 residues in a 1:1 equimolar complex with substrate-free cytP450. Green, cyan, and magenta highlight the extensive peak broadening observed for cytb5 residues upon addition of the increasing amounts of unlabeled cytP450 bound to BHT (A = 1:0.3, B = 1:0.6, and C = 1:1 molar ratios between cytb5 and cytP450). All peak intensities were normalized to the C-terminal residue Asp-134 in the unbound cytb5 spectrum to account for the change in intensity upon complex formation. B and C present two different views of cytb5 rotated by 90° with respect to each other and a space-filling representation of the second view. B, cytb5 residues exhibiting extensive line broadening (with a decrease in peak height >20% as compared with free cytb5) upon complex formation with an equimolar amount of substrate/ligand-free cytP450 are colored orange onto the NMR structure of cytb5. C, residues of cytb5 whose resonances are broadened beyond detection upon complex formation with an equimolar amount of cytP450 bound to BHT are represented in magenta. All NMR data were collected on the full-length complex incorporated in isotropic bicelles.
      A histogram depicting the weighted average chemical shift perturbations (Δδ) observed for residues of cytb5 upon complex formation with cytP450 is presented in Fig. 5. The average chemical shift perturbations observed for the backbone amides of cytb5, upon addition of cytP450, are relatively small in magnitude, <0.01 ppm, and are spread over a large area of cytb5. As a result, no specific regions of cytb5 can be highlighted as being part of the interaction epitope, based on chemical shift perturbations. This lack of widespread changes in chemical shifts across the 1H-15N TROSY-HSQC spectrum indicates that there is no notable change in the overall tertiary fold of cytb5 upon interaction with cytP450.
      The reason for the small average chemical shift perturbations could be 2-fold. First, fast-to-intermediate (ns-μs) chemical exchange between the free observable cytb5 in isotropic bicelles and the unobservable high molecular weight bound-state of the cytb5-cytP450 complex in isotropic bicelles (> 100 kDa) (
      • Prudêncio M.
      • Ubbink M.
      Transient complexes of redox proteins: structural and dynamic details from NMR studies.
      ,
      • Zuiderweg E.R.
      Mapping protein-protein interactions in solution by NMR spectroscopy.
      ) would explain the overall broadening of the observed cytb5 resonances. Our findings are in agreement with a recent NMR study, where they observed very modest chemical shift perturbations upon complex formation between truncated forms of cytP45017A1 and human cytb5 lacking the membrane domains (
      • Estrada D.F.
      • Laurence J.S.
      • Scott E.E.
      Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.
      ). Second, small and widespread chemical shift perturbations could be a result of the formation of an ensemble of dynamic “encounter complexes” as have been reported previously for other metalloprotein complexes (
      • Prudêncio M.
      • Ubbink M.
      Transient complexes of redox proteins: structural and dynamic details from NMR studies.
      ,
      • Volkov A.N.
      • Ferrari D.
      • Worrall J.A.
      • Bonvin A.M.
      • Ubbink M.
      The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.
      ,
      • Tang C.
      • Iwahara J.
      • Clore G.M.
      Visualization of transient encounter complexes in protein-protein association.
      ,
      • Volkov A.N.
      • Ubbink M.
      • van Nuland N.A.
      Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy.
      ,
      • Suh J.Y.
      • Tang C.
      • Clore G.M.
      Role of electrostatic interactions in transient encounter complexes in protein-protein association investigated by paramagnetic relaxation enhancement.
      ) such as cytb5-myoglobin (
      • Prudêncio M.
      • Ubbink M.
      Transient complexes of redox proteins: structural and dynamic details from NMR studies.
      ,
      • Zuiderweg E.R.
      Mapping protein-protein interactions in solution by NMR spectroscopy.
      ). Encounter complexes are composed of an ensemble of protein orientations within the complex; this leads to very small chemical shift perturbations as observed for the cytb5-cytP450 complex here and for other redox partners previously (
      • Prudêncio M.
      • Ubbink M.
      Transient complexes of redox proteins: structural and dynamic details from NMR studies.
      ).
      A closer inspection of Fig. 6A reveals differential line broadening of cytb5 resonances upon complex formation with substrate-free cytP450 (yellow). The line broadening could be the result of the following: (a) changes in chemical shifts suggesting a conformational change in the protein or (b) a change in the transverse relaxation rate of cytb5 resonances caused by a direct interaction with cytP450 (
      • Matsuo H.
      • Walters K.J.
      • Teruya K.
      • Tanaka T.
      • Gassner G.T.
      • Lippard S.J.
      • Kyogoku Y.
      • Wagner G.
      Identification by NMR spectroscopy of residues at contact surfaces in large, slowly exchanging macromolecular complexes.
      ). The absence of significant chemical shift perturbations (as mentioned above) suggests that the differential line broadening (Fig. 6) observed is predominantly due to a direct interaction with cytP450, enabling the characterization of the interaction interface between cytb5 and cytP450 (
      • Matsuo H.
      • Walters K.J.
      • Teruya K.
      • Tanaka T.
      • Gassner G.T.
      • Lippard S.J.
      • Kyogoku Y.
      • Wagner G.
      Identification by NMR spectroscopy of residues at contact surfaces in large, slowly exchanging macromolecular complexes.
      ,
      • Zamoon J.
      • Nitu F.
      • Karim C.
      • Thomas D.D.
      • Veglia G.
      Mapping the interaction surface of a membrane protein: unveiling the conformational switch of phospholamban in calcium pump regulation.
      ). cytb5 residues that exhibited significant differential line broadening (with a decrease in peak height of greater than 20% as compared with free cytb5) were mapped onto the NMR structure of cytb5. These residues, which include Glu-48, Glu-49, Asp-65, Val-66, and Thr-70 to Ser-76, highlight a region of cytb5 around the solvent-exposed edge of the heme that potentially forms the interaction interface with cytP450 (Fig. 6B). Broadening of His-68 and Ser-69 resonances of cytb5 was also observed and may be due to the close proximity of the paramagnetic center in cytP450 and/or due to steric interaction between His-68 and Ser-69 and residues on cytP450 in the interface. We also observed broadening of the backbone amide-NH resonances corresponding to residues Met-96 to Val-103 in the flexible linker domain of cytb5 (Fig. 5); this observed broadening is likely due to restriction of the motion of the linker upon complex formation (
      • Koberova M.
      • Jecmen T.
      • Sulc M.
      • Cerna V.
      • Kizek R.
      • Stiborova M.
      • Hodek P.
      Photo-cytochrome b5–a new tool to study the cytochrome P450 electron-transport chain.
      ).
      Interestingly, extensive line broadening and disappearance of most of the cytb5 amide resonances in the 1H-15N TROSY-HSQC spectrum were observed upon addition of an equimolar amount of substrate-bound unlabeled cytP450 (Fig. 6C). Two different compounds were tested as follows: BHT (type I substrate), and the heme iron-binding inhibitor, 1-(4-chlorophenyl)imidazole (1-CPI; type II ligand).
      Type I ligands displace the water coordinating the Fe(III) in the heme as the sixth ligand, shifting the Fe(III) spin equilibrium toward the high spin form, whereas type II ligands can replace the water by coordinating to the Fe(III) thereby stabilizing the low spin form.
      The widespread broadening of the cytb5 resonances might suggest that the interaction of cytb5 with substrate-bound cytP450 has shifted from a fast-to-intermediate (nanosecond to microsecond) to an intermediate-to-slow time scale (microsecond to millisecond), which causes the disappearance of the majority of cytb5 resonances upon titration of substrate-bound cytP450. This conjecture is further supported by the measurement of a submicromolar Kd value for the cytb5-cytP450 complex in the presence of the substrates methoxyflurane (Kd ∼0.02 μm) (Table 3), BHT (Kd ∼ 0.3 μm) (data not shown), and 1-CPI (Kd ∼ 0.03 μm) (data not shown) in aqueous solution. These submicromolar Kd values are consistent with an intermediate-to-slow exchange on the NMR time scale leading to extensive line broadening of cytb5 amide resonances (
      • Zuiderweg E.R.
      Mapping protein-protein interactions in solution by NMR spectroscopy.
      ). A previous kinetic study has also reported a greater than 10-fold decrease in Kd of the cytb5-cytP450 complex upon addition of the substrate, benzphetamine (
      • Tamburini P.P.
      • Gibson G.G.
      Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein.
      ). However, due to extensive line broadening and disappearance of most of the resonances of the heme domain of cytb5 in the 1H-15N TROSY-HSQC spectrum, relaxation NMR experiments could not be performed to validate the change in the time scale of interaction.

      Mutagenesis Identifies Residues in Contact in the cytb5-cytP450 Complex

      To complement the NMR data collected on the cytb5-cytP450 complex, we carried out site-directed mutagenesis of residues on both cytb5 and cytP450 (supplemental Fig. S2B and TABLE 3, TABLE 4). Residues (Glu-42, Glu-43, Pro-45, Gly-46, Glu-49, Val-50, Glu-53, Gln-54, Asn-62, Asp-65, Val-66, Asp-71, and Leu-75) on the anionic surface surrounding the solvent-exposed heme of cytb5 were mutated to alanine to explore the role of atoms distal to the β-carbon of the wild-type amino acid in binding to cytP4502B4 (supplemental Fig. S2B). After purification, the mutant proteins were assayed for their ability to bind cytP4502B4 and stimulate catalysis in an aqueous reconstituted system (Table 3). Of the 13 different single mutations of cytb5, only two, D65A and V66A, exhibited both a significantly lower affinity for cytP4502B4 (15- and 7-fold higher Kd, respectively) and a decreased ability (85 and 43%, respectively) to stimulate cytP4502B4 catalysis (Table 3). These data indicate that Asp-65 and Val-66 of cytb5 are important for both binding to cytP450 and its function as an enhancer of cytP450 catalysis. Site-directed mutants of cytb5, P45A, G46A, E53A, Q54A, D71A, and L75A were found to be indistinguishable from wild type (data not shown). Whereas E42A, E43A, E49A, V50A, and N62A exhibited a modest decrease in binding affinity to cytP450, these mutants did not show a decrease in their ability to stimulate cytP4502B4 activity (Table 3); as a result, these residues were deemed to only play a minor role in the interprotein interactions.
      TABLE 4Double mutant cycle analysis of mutants of cytP450 and cytb5
      Difference in free energy of binding (kcal/mol)
      cytP450cytb5Kdm)
      a All Kd values were measured in the presence of methoxyflurane.
      cytb5-cytP450
      Free energy of binding (kcal/mol) ΔG
      b Free energy change of binding of the indicated cytP450 and cytb5 is shown.
      cytb5-cytP450
      ΔΔ[vi]G[v]
      c The difference in free energy of binding between a complex containing a single mutant protein and a complex with two wild-type proteins is shown: ΔΔG = ΔGmutant − ΔGwild type.
      [v] of mutant-wild-type
      ΔΔG
      d The free energy of interaction between the two mutant proteins is shown: ΔΔGinteraction of mutants = ΔΔGmutant cytP450-wild-type cytb5 + ΔΔGwild-type cytP450-mutant cytb5 − ΔΔGmutant cytp450-mutant cytb5.
      interaction of double mutants
      Wild typeWild type0.022 ± 0.003−10.43
      R122AWild type0.221 ± 0.010−9.071.36
      Wild typeD65A0.332 ± 0.032−8.821.61
      R122AD65A0.558 ± 0.065−8.521.911.06
      Wild typeV66A0.152 ± 0.019−9.291.14
      R122AV66A0.814 ± 0.033−8.292.140.37
      R126AWild type0.454 ± 0.042−8.641.79
      Wild typeD65A0.332 ± 0.042−8.821.61
      R126AD65A18.33 ± 1.500−6.453.98−0.58
      Wild typeV66A0.152 ± 0.010−9.291.14
      R126AV66A5.149 ± 0.930−7.203.23−0.30
      F135AWild type0.205 ± 0.021−9.111.32
      Wild typeD65A0.332 ± 0.042−8.821.61
      F135AD65A1.420 ± 0.110−7.962.470.46
      Wild typeV66A0.152 ± 0.010−9.291.14
      F135AV66A0.959 ± 0.130−8.202.230.23
      M137AWild type0.379 ± 0.040−8.751.68
      Wild typeD65A0.332 ± 0.012−8.821.61
      M137AD65A3.951 ± 0.440−7.363.070.22
      Wild typeV66A0.152 ± 0.010−9.291.14
      M137AV66A1.583 ± 0.142−7.902.530.30
      K139AWild type0.611 ± 0.050−8.461.97
      Wild typeD65A0.332 ± 0.012−8.821.61
      K139AD65A4.831 ± 0.042−7.243.190.38
      Wild typeV66A0.152 ± 0.010−9.291.14
      K139AV66A1.683 ± 0.212−7.862.570.52
      K433AWild type0.458 ± 0.053−8.631.80
      Wild typeD65A0.332 ± 0.012−8.821.61
      K433AD65A0.869 ± 0.071−8.262.17
      Wild typeV66A0.152 ± 0.010−9.291.14
      K433AV66A0.487 ± 0.052−8.601.83
      a a All Kd values were measured in the presence of methoxyflurane.
      b b Free energy change of binding of the indicated cytP450 and cytb5 is shown.
      c c The difference in free energy of binding between a complex containing a single mutant protein and a complex with two wild-type proteins is shown: ΔΔG = ΔGmutant − ΔGwild type.
      d d The free energy of interaction between the two mutant proteins is shown: ΔΔGinteraction of mutants = ΔΔGmutant cytP450-wild-type cytb5 + ΔΔGwild-type cytP450-mutant cytb5 − ΔΔGmutant cytp450-mutant cytb5.
      Previous mutagenesis studies on cytP4502B4 have shown that residues in the C-helix and C-D loop, which were mutated to alanine (Arg-122, Arg-126, Arg-133, Phe-135, Met-137, and Lys-139) and Lys-433 in the β-bulge near the axial Cys-436, are important for binding to cytb5 (Table 3) (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). The R133A mutant showed a drastic decrease in binding affinity to cytb5; in fact, the binding was too weak to obtain a robust Kd measurement. All other mutants showed a decrease in binding affinity of at least 10-fold.
      To determine the amino acids that are in contact at the interface between cytb5 and cytP450, a “double mutant cycle” (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ,
      • Frisch C.
      • Schreiber G.
      • Johnson C.M.
      • Fersht A.R.
      Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.
      ) analysis was then performed using mutants of both cytb5 and cytP450 that are defective in binding to one another (Table 4). In such cycles, the sum of the free energy change for the two single amino acid mutant proteins is compared with that of the double mutant protein complex. When the sum of the free energy change of the single mutants is not equal to that of the double mutant, the two residues are defined as interacting and not behaving independently. This assumes that the two residues are not interacting indirectly, e.g. through a structural perturbation (
      • Frisch C.
      • Schreiber G.
      • Johnson C.M.
      • Fersht A.R.
      Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.
      ,
      • Harel M.
      • Cohen M.
      • Schreiber G.
      On the dynamic nature of the transition state for protein-protein association as determined by double-mutant cycle analysis and simulation.
      ). A difference of greater than 1.0 kcal/mol was considered significant (
      • Frisch C.
      • Schreiber G.
      • Johnson C.M.
      • Fersht A.R.
      Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.
      ,
      • Harel M.
      • Cohen M.
      • Schreiber G.
      On the dynamic nature of the transition state for protein-protein association as determined by double-mutant cycle analysis and simulation.
      ). The free energy of binding, ΔG, of all possible pairs of wild type and poorly binding alanine mutants (Table 3) of both cytP4502B4 (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ) (Arg-122, Arg-126, Phe-135, Met-137, Lys-139, and Lys-433) and cytb5 (Asp-65 and Val-66) was measured. Table 4 presents the results of the double mutant cycle analysis, which indicate that Lys-433 of cytP450 interacts with both Asp-65 and Val-66 of cytb5 and that Arg-122 of cytP450 interacts with Asp-65 of cytb5. Because of the 68-fold decreased affinity of the R133A-cytP450 mutant for cytb5, a robust double mutant cycle analysis could not be performed (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ); however, R133A-cytP450 mutant's poor affinity for cytb5 indicates that Arg-133 is critical to the interprotein interaction.

      Structure of the cytb5-cytP450 Complex

      A structural model of the cytb5-cytP450 complex was generated using the data-driven docking program HADDOCK (
      • de Vries S.J.
      • van Dijk A.D.
      • Krzeminski M.
      • van Dijk M.
      • Thureau A.
      • Hsu V.
      • Wassenaar T.
      • Bonvin A.M.
      HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
      ), governed by unambiguous and ambiguous intermolecular restraints obtained from mutagenesis and NMR data (Table 5). The solution NMR structure of the heme domain of the membrane-bound rabbit cytb5 with a backbone r.m.s.d. of 0.32 ± 0.10 Å (Fig. 1A) and a 1.9 Å resolution crystal structure of the heme-containing domain of cytP4502B4 (PDB code 1SUO (
      • Scott E.E.
      • White M.A.
      • He Y.A.
      • Johnson E.F.
      • Stout C.D.
      • Halpert J.R.
      Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
      )) were used in HADDOCK calculations. Docking was performed in the absence of a membrane environment, and therefore our structure of the complex reveals the interactions between the structured heme domains of cytb5 and cytP450. However, it is important to note that all NMR and mutagenesis data were collected on membrane-bound full-length proteins, and the enzymatic function of the complex under these conditions was confirmed by activity assays (supplemental Table S1).
      As described above, the double mutant cycle analysis revealed that Lys-433 of cytP450 interacts with both Asp-65 and Val-66 of cytb5 and that Arg-122 of cytP450 interacts with Asp-65 of cytb5; these interactions were incorporated as unambiguous intermolecular restraints in HADDOCK. The ambiguous intermolecular restraints were generated using active and passive residues for cytb5 and cytP450 (all >40% solvent-accessible). As active residues, eight cytb5 residues exhibiting significant differential line broadening upon complex formation with cytP450, and seven cytP450 residues deemed essential for binding to cytb5, based on site-directed mutagenesis, were selected. As passive residues, solvent-accessible (>40% solvent-accessible) amino acids flanking the active residues were selected for cytb5, and for cytP450, all residues on the proximal side where the heme is closest to the surface were selected (Table 5).
      The docking simulations reveal not a single specific complex but rather an ensemble of low energy complex orientations (supplemental Fig. S3A), where the acidic convex surface of cytb5 is sampling an extended surface area on the concave basic proximal side of cytP450 (data not shown). The two dominant subpopulations of low energy complexes (Table 6 and supplemental Fig. S3), titled clusters I and II, include two unique but overlapping clusters of residues on cytb5 and cytP4502B4 (FIGURE 7, FIGURE 8, supplemental Figs S2 and S3, and supplemental Table S2). The residues of cytb5 that are common between the majority of the low energy complex structures (supplemental Fig. S3) are found mostly on the α4 and α5 helices). The cytP450 and cytb5 hemes are nearly perpendicular to one another in both clusters, and the shortest distance between the two heme edges is 9.0 and 7.4 Å, respectively, in clusters I and II, which is well within the 14.0 Å limit predicted for efficient electron transfer (Fig. 7B) (
      • Page C.C.
      • Moser C.C.
      • Dutton P.L.
      Mechanism for electron transfer within and between proteins.
      ). The Fe-Fe distance is 20.9 and 19.3 Å in clusters I and II, respectively.
      TABLE 6Energy statistics for the two lowest energy clusters of the complex between cytb5 and cytP450 generated from HADDOCK
      ParametersCluster ICluster II
      No. of structures from the 50 lowest energy -docked solutions2114
      Backbone r.m.s.d. (as compared with the reference structure)0.81 ± 0.29 Å2.67 ± 0.42 Å
      Total energy−435.8 ± 38.0 kcal/mol−470.7 ± 49.7 kcal/mol
      van der Waals energy−43.8 ± 8.0 kcal/mol−29.5 ± 7.2 kcal/mol
      Electrostatic energy−392.0 ± 37.2 kcal/mol−441.2 ± 53.7 kcal/mol
      Desolvation energy33.9 ± 3.0 kcal/mol35.8 ± 3.6 kcal/mol
      Interface surface area937.0 ± 88.0 Å2903.1 ± 77.7 Å2
      Figure thumbnail gr8
      FIGURE 8Binding interface of the membrane-bound cytb5-cytP450 complex. The complex is presented by opening the complex-like pages of a book with the interaction interface of cytb5 and cytP450 facing the viewer. The space-filling model of cytb5 (NMR structure) and cytP450 (PDB code 1SUO (
      • Scott E.E.
      • White M.A.
      • He Y.A.
      • Johnson E.F.
      • Stout C.D.
      • Halpert J.R.
      Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
      )) is presented highlighting the interfacial residues involved in protein-protein contacts in cluster I (A) and cluster II (B) complex structures. Residues on cytb5 that are in contact with residues on cytP450 are denoted with matching letters in parentheses. For example, Asp-65 (orange) on cytb5 is H-bonding to Arg-122 (blue) on cytP450 in A. Arg-125 highlighted in blue is H-bonded to the heme-d-propionate in B. An important point to note is that the residues on cytb5 and cytP450, which form the interaction interface, are largely the same between the two clusters and are mostly present on the lower edge (residues on α4 and α5 helix) of the soluble domain surrounding the heme. The residues in the interface are in excellent agreement with our NMR data and site-directed mutagenesis presented here (TABLE 3, TABLE 4), as well as elsewhere (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ).
      Figure thumbnail gr7
      FIGURE 7Structure of the full-length membrane-bound cytb5-cytP450 complex. A, two lowest energy clusters (I and II) of the complex between the catalytic heme-binding domains of rabbit cytb5 (NMR structure; blue) and cytP4502B4 (PDB code 1SUO (
      • Scott E.E.
      • White M.A.
      • He Y.A.
      • Johnson E.F.
      • Stout C.D.
      • Halpert J.R.
      Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
      ); in gold) generated from HADDOCK, driven by NMR, and mutagenesis restraints. Heme molecules are presented in red. B, proposed electron transfer pathway between the redox centers of cytb5 and cytP450 are presented as broken lines. The shortest electron transfer pathway predicted using HARLEM (
      • Kurnikov I.V.
      ) is shown in the black dotted lines for clusters I and II. The shortest heme-edge to heme-edge distance is 7.4 and 9.0 Å in clusters II and I, respectively.

      DISCUSSION

      The structure of the heme domain of full-length rabbit cytb5, incorporated in DPC micelles, was found to be similar but not identical to the previously reported structure of truncated rabbit cytb5 (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ). Our NMR structure has additional β-strands (β1 and β4) and longer (α1, β2, α2, and α3) and shorter (β5 and 310 helix) segments for some of the secondary structure elements (FIGURE 1, FIGURE 3 and supplemental Fig. S1A). The linker region (Ser-90 to Asp-104) in our NMR structure of cytb5, which connects the cytosolic heme domain to the α-helical transmembrane anchor, was found to be random coil. As mentioned under “Results,” the extended form of the cytb5 linker region likely allows for proper interaction of the cytb5 soluble domain with its redox partners. Solid-state SLF NMR data, in combination with a structure fitting algorithm (
      • Nevzorov A.A.
      • Opella S.J.
      Structural fitting of PISEMA spectra of aligned proteins.
      ), were used to determine the α-helical structure of the transmembrane domain of cytb5 (Fig. 1C). Our NMR structure of full-length microsomal ferric cytb5 was used subsequently to establish the interaction interface between microsomal, rabbit, ferric cytb5, and cytP450.

      cytb5-cytP450 Interaction Interface

      As mentioned above, the modest average chemical shift perturbations (Δδ <0.01 ppm) observed for the heme domain of full-length cytb5 upon complex formation with substrate-free cytP450 could be a result of the combination of interaction on the fast-to-intermediate NMR exchange time scale and formation of an ensemble of dynamic encounter complexes, as has been reported previously for other metalloprotein complexes (
      • Volkov A.N.
      • Ferrari D.
      • Worrall J.A.
      • Bonvin A.M.
      • Ubbink M.
      The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.
      ,
      • Suh J.Y.
      • Tang C.
      • Clore G.M.
      Role of electrostatic interactions in transient encounter complexes in protein-protein association investigated by paramagnetic relaxation enhancement.
      ). Complex formation between electron transfer proteins, like cytP450 and cytb5, has been shown to proceed via the formation of dynamic encounter complexes driven by the oppositely charged surfaces of the proteins (supplemental Fig. S2A) (
      • Suh J.Y.
      • Tang C.
      • Clore G.M.
      Role of electrostatic interactions in transient encounter complexes in protein-protein association investigated by paramagnetic relaxation enhancement.
      ,
      • Ubbink M.
      The courtship of proteins: Understanding the encounter complex.
      ). In our experiment, we hypothesize that the different complex orientations, within the encounter complexes, are interchanging among themselves at a fast-to-intermediate time scale, because we do not see any significant chemical shift perturbations. These encounter complexes formed by cytb5 and cytP450 in the absence of a substrate are most likely in equilibrium with a well defined complex orientation, known as the stereospecific complex (
      • Ubbink M.
      The courtship of proteins: Understanding the encounter complex.
      ). The stereospecific complex is characterized by a tighter affinity of the two proteins for one another and more hydrophobic interactions, whereas the weaker encounter complexes are stabilized predominantly by long range electrostatic interactions. The lifetime and the populations of the individual orientations govern the effect of the encounter and the stereospecific complex on the NMR data.
      The extensive line broadening of cytb5 resonances upon complex formation with substrate-bound cytP450 (Fig. 6) suggests that substrate binding modulates the affinity of the proteins for each other. Our result is in agreement with two recent studies where binding of the substrate modulates the interaction between cytP450 and cytb5 (
      • Estrada D.F.
      • Laurence J.S.
      • Scott E.E.
      Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.
      ,
      • Koberova M.
      • Jecmen T.
      • Sulc M.
      • Cerna V.
      • Kizek R.
      • Stiborova M.
      • Hodek P.
      Photo-cytochrome b5–a new tool to study the cytochrome P450 electron-transport chain.
      ). Addition of the substrate could be shifting the equilibrium from the weaker encounter complexes toward the stereospecific complex.
      The two lowest energy cytb5-cytP450 complex orientations (clusters I and II), calculated from HADDOCK driven by the NMR and mutagenesis data presented here, likely represent “productive” cytb5-cytP450 complex orientations, because the heme-edge to heme-edge distances would allow for efficient electron transfer (
      • Page C.C.
      • Moser C.C.
      • Dutton P.L.
      Mechanism for electron transfer within and between proteins.
      ). Both complex structures are typical of other redox complexes in that, although there is a large interfacial area of contact (∼937 Å2 in cluster I and ∼903 Å2 in cluster II, see Table 6), the bulk of the binding energy can be attributed to a small number of complementary residues (
      • Clackson T.
      • Wells J.A.
      A hot spot of binding energy in a hormone-receptor interface.
      ). Our result is in agreement with a recent chemical cross-linking study on cytP4502B4 where the authors observed evidence for the existence of two mutual orientations of the cytb5-cytP450 complex (
      • Sulc M.
      • Jecmen T.
      • Snajdrova R.
      • Novak P.
      • Martinek V.
      • Hodek P.
      • Stiborova M.
      • Hudecek J.
      Mapping of interaction between cytochrome P450 2B4 and cytochrome b5: the first evidence of two mutual orientations.
      ).
      The cytb5-cytP450 complex orientations reveal that the acidic convex surface of cytb5 docks, like a ball in a socket, into the entire concavity on the proximal surface of cytP450, with the C-helix residues contributing the vast majority of the binding energy as indicated by the mutagenesis data (Table 3). A closer look at the complex structures from clusters I and II (Fig. 8, supplemental Fig. S2, C and D, and supplemental Table S2) highlights that the interactions at the complex interface occur between 14 residues and the heme-d-propionate of cytb5 and 14 residues of cytP450. Based on the double mutant cycle analysis (Table 4), we identified that the interactions contributing the most binding energy to the complex formation were between the cytP450 C-helix residue Arg-122 and Lys-433 in the β-bulge (near the axial Cys-436) and Asp-65 and Val-66 at the C terminus of helix α4 of cytb5. These interactions were shown to be critical for both complex formation and function. From the HADDOCK structures, we see that Asp-65 of cytb5 is able to form hydrogen bonds and/or salt bridges with Arg-122 and Lys-433 of cytP450 and that Val-66 of cytb5 is in van der Waals contact with Lys-433 (and Arg-125) of cytP450. Arg-133 of cytP450, which was found to be very important for binding to cytb5 in our studies, is hydrogen-bonded with the heme propionate group on cytb5 in both clusters (additional interactions were also found for Arg-133 in cluster II, see supplemental Table S2). The function of other cytP4502B4 residues previously mutated to alanine (Table 3) (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ) can also be discussed. Arg-126 of cytP450 was found to form hydrogen bonds and salt bridges with Glu-64 of cytb5 in both clusters and hydrogen bonds with His-68 (supplemental Table S2). Surprisingly, the C-D loop residues of cytP450, Met-137, and Lys-139, which mutagenesis data revealed were important for cytb5 binding (Table 3), were not in the complex interface (Fig. 8 and supplemental Fig. S2, C and D). We hypothesize that their mutation might induce a structural perturbation in the flexible C-helix, which in turn destabilizes the interaction with cytb5. The K139A mutation was previously shown to disrupt a hydrogen bond network between the Lys-139 amino group and Pro-261 and Asn-260 in the G-H loop (
      • Scott E.E.
      • White M.A.
      • He Y.A.
      • Johnson E.F.
      • Stout C.D.
      • Halpert J.R.
      Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
      ), suggesting an allosteric interaction between the C-helix and the G-H loop. Arg-422 on cytP450, which is in contact with cytb5 in the predicted complex structure from cluster II, was previously considered to be important only for binding to CPR (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). However, review of the previously reported mutagenesis data revealed that the R422A-cytP450 mutant exhibited a 50% decreased affinity for cytb5, which at the time was considered insignificant (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ). Residue Arg-443, which was shown to be important only for cytP450 reductase (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ), is not found in the interaction interface of the predicted complex structures (Fig. 8).
      On cytb5, both Asp-65 and Val-66, which were shown to be in the binding site for cytochrome c (
      • Banci L.
      • Bertini I.
      • Rosato A.
      • Scacchieri S.
      Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
      ,
      • Shao W.
      • Im S.C.
      • Zuiderweg E.R.
      • Waskell L.
      Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance.
      ), are now shown to be in the binding site for cytP4502B4 both experimentally and in our HADDOCK complex structures. A double cytb5 mutant E48G and E49G mutant (which introduced a very flexible sequence of four glycine residues) has been shown to be deficient in its ability to stimulate the activity of cytP450c17 (
      • Naffin-Olivos J.L.
      • Auchus R.J.
      Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17.
      ). This observation is consistent with the presence of Glu-48 and Glu-49 in the interaction interface of cluster II and is in accordance with our NMR data where we observed considerable line broadening for Glu-48 and Glu-49 upon complex formation with cytP450.
      The HADDOCK structures of the cytb5-cytP450 complex, as well as previous experiments (
      • Zhang H.
      • Hamdane D.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4.
      ,
      • Zhang H.
      • Im S.C.
      • Waskell L.
      Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.
      ), demonstrate that cytb5 and CPR compete for an overlapping binding site on cytP450, and they rule out the existence of separate functional binding sites for cytb5 and CPR and the formation of a ternary complex between the three proteins (
      • Tamburini P.P.
      • Schenkman J.B.
      Purification to homogeneity and enzymological characterization of a functional covalent complex composed of cytochromes P-450 isozyme 2 and b5 from rabbit liver.
      ).

      Electron Transfer Pathway between cytb5 and cytP450

      The structure of the cytb5-cytP450 complex generated from HADDOCK shows that the guanidinium group of Arg-125 on the C-helix of cytP450 forms a salt bridge between the heme-d-propionates of both cytb5 and cytP450. This network was predicted, using HARLEM (
      • Kurnikov I.V.
      ), to serve as one of the shortest electron transfer pathways between the two proteins (Fig. 7B). Arg-125 is one of the most highly conserved cytP450 residues. It is homologous to Arg-112 in cytP450cam, which has been shown to be essential for electron transfer (
      • Nakamura K.
      • Horiuchi T.
      • Yasukochi T.
      • Sekimizu K.
      • Hara T.
      • Sagara Y.
      Significant contribution of arginine 112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin.
      ). The physiological significance of Arg-125 was also highlighted when mutation of the Arg-125 homolog in human cytP45024A1 resulted in a defect in vitamin D degradation (
      • Schlingmann K.P.
      • Kaufmann M.
      • Weber S.
      • Irwin A.
      • Goos C.
      • John U.
      • Misselwitz J.
      • Klaus G.
      • Kuwertz-Bröking E.
      • Fehrenbach H.
      • Wingen A.M.
      • Güran T.
      • Hoenderop J.G.
      • Bindels R.J.
      • Prosser D.E.
      • Jones G.
      • Konrad M.
      Mutations in CYP24A1 and idiopathic infantile hypercalcemia.
      ). We have previously attempted to characterize the R125A mutant of cytP4502B4 (
      • Bridges A.
      • Gruenke L.
      • Chang Y.T.
      • Vakser I.A.
      • Loew G.
      • Waskell L.
      Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
      ), and we found that the mutation rendered the protein unstable. The proposed network involving the heme propionates is consistent with previous studies that have demonstrated that the heme propionate groups of cytb5 interact with charged groups on cytochrome c (
      • Rodgers K.K.
      • Pochapsky T.C.
      • Sligar S.G.
      Probing the mechanisms of macromolecular recognition-the cytochrome-b5-cytochrome c complex.
      ,
      • Deep S.
      • Im S.C.
      • Zuiderweg E.R.
      • Waskell L.
      Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data.
      ) and cytP450 isozymes (
      • Tamburini P.P.
      • Schenkman J.B.
      Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups.
      ), suggesting that other cytP450 complexes employ a similar interface. Fig. 7B presents another possible electron transfer pathway between the heme-d-propionate of cytb5 and Ile-435, the nonconserved amino acid preceding the axial ligand, Cys-436, on cytP450. However, our mutagenesis data have shown that the I435A mutant is as active as the wild-type cytP450 (data not shown), suggesting that it does not play a critical role in electron transfer between the two proteins.

      Conclusion

      Here, we have presented the first full-length structure of rabbit cytb5 incorporated in a membrane mimetic (DPC micelles or lipid bicelles) obtained using a combination of solution and solid-state NMR spectroscopy. The heme domain and linker region structures were established using solution NMR, and the transmembrane domain structure and topology were identified using solid-state NMR (full-length cytb5 was used for all NMR measurements). The highly conserved nature of the transmembrane domain of cytb5 (with a sequence similarity of 78–96% (
      • Clarke T.A.
      • Im S.C.
      • Bidwai A.
      • Waskell L.
      The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
      ) among vertebrates) suggests that these results and methodology should be applicable to other mammalian cytsb5 as well.
      Subsequently, HADDOCK (
      • de Vries S.J.
      • van Dijk A.D.
      • Krzeminski M.
      • van Dijk M.
      • Thureau A.
      • Hsu V.
      • Wassenaar T.
      • Bonvin A.M.
      HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
      ), driven by experimental constraints obtained from site-directed mutagenesis and solution NMR spectroscopy on full-length membrane-bound microsomal cytb5 and cytP4502B4, was used to generate a model of the cytb5-cytP450 complex. The two proteins form a dynamic complex mediated by both hydrophobic and electrostatic interactions (Fig. 8 and supplemental Table S2). The electrostatic interactions between the oppositely charged residues, as well as the fact that the two proteins are anchored in the membrane, play important roles in orienting the two proteins prior to complex formation and help considerably by increasing the number of productive collisions that control and direct the flow of electrons from cytb5 to cytP450. Addition of a small molecule substrate (BHT) or inhibitor (1-CPI) significantly increased the binding affinity between cytb5 and cytP450, moving the dynamic interaction between the two proteins from a fast-to-intermediate regime to an intermediate-to-slow regime on the NMR time scale, based on the extensive line broadening of cytb5 amide NMR resonances upon complex formation with substrate-bound cytP450. The structure of the cytb5-cytP450 complex presented allows us to identify the interactions at the interface and to propose the pathway of interprotein electron transfer from cytb5 to cytP450 through the highly conserved Arg-125 residue on cytP450. Our study demonstrates how a combinatorial approach, involving NMR and mutagenesis studies, can be exploited to obtain atomic level structural, functional, and dynamic information on intact, membrane-bound, and large metalloprotein redox complexes in a near-native environment. The extensive knowledge of the structure of the cytb5-cytP450 complex provides insights into the principles governing interprotein interactions and will markedly facilitate our ability to unravel the molecular mechanism by which the rate of cytP450 catalysis is regulated by its redox partners, cytb5 and CPR.

      REFERENCES

        • Guengerich F.P.
        • Wu Z.L.
        • Bartleson C.J.
        Function of human cytochrome P450s: characterization of the orphans.
        Biochem. Biophys. Res. Commun. 2005; 338: 465-469
        • Shen A.L.
        • O'Leary K.A.
        • Kasper C.B.
        Association of multiple developmental defects and embryonic lethality with loss of microsomal NADPH-cytochrome p450 oxidoreductase.
        J. Biol. Chem. 2002; 277: 6536-6541
        • Nebert D.W.
        • Russell D.W.
        Clinical importance of the cytochromes P450.
        Lancet. 2002; 360: 1155-1162
        • O'Donnell A.
        • Judson I.
        • Dowsett M.
        • Raynaud F.
        • Dearnaley D.
        • Mason M.
        • Harland S.
        • Robbins A.
        • Halbert G.
        • Nutley B.
        • Jarman M.
        Hormonal impact of the 17α-hydroxylase/C(17,20)-lyase inhibitor abiraterone acetate (CB7630) in patients with prostate cancer.
        Br. J. Cancer. 2004; 90: 2317-2325
        • Orlando L.
        • Schiavone P.
        • Fedele P.
        • Calvani N.
        • Nacci A.
        • Rizzo P.
        • Marino A.
        • D'Amico M.
        • Sponziello F.
        • Mazzoni E.
        • Cinefra M.
        • Fazio N.
        • Maiello E.
        • Silvestris N.
        • Colucci G.
        • Cinieri S.
        Molecularly targeted endocrine therapies for breast cancer.
        Cancer Treat. Rev. 2010; 36: S67-S71
        • Im S.C.
        • Waskell L.
        The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase, and cytochrome b5.
        Arch. Biochem. Biophys. 2011; 507: 144-153
        • Zhang H.
        • Hamdane D.
        • Im S.C.
        • Waskell L.
        Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4.
        J. Biol. Chem. 2008; 283: 5217-5225
        • Gruenke L.D.
        • Konopka K.
        • Cadieu M.
        • Waskell L.
        The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.
        J. Biol. Chem. 1995; 270: 24707-24718
        • Finn R.D.
        • McLaughlin L.A.
        • Ronseaux S.
        • Rosewell I.
        • Houston J.B.
        • Henderson C.J.
        • Wolf C.R.
        Defining the in vivo role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.
        J. Biol. Chem. 2008; 283: 31385-31393
        • Guengerich F.P.
        Cytochrome P450s and other enzymes in drug metabolism and toxicity.
        AAPS J. 2006; 8: E101-E111
        • Shimada T.
        • Mernaugh R.L.
        • Guengerich F.P.
        Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b5 enzymes.
        Arch. Biochem. Biophys. 2005; 435: 207-216
        • Canova-Davis E.
        • Chiang J.Y.
        • Waskell L.
        Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.
        Biochem. Pharmacol. 1985; 34: 1907-1912
        • Morgan E.T.
        • Coon M.J.
        Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5.
        Drug Metab. Dispos. 1984; 12: 358-364
        • Zhang H.
        • Im S.C.
        • Waskell L.
        Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.
        J. Biol. Chem. 2007; 282: 29766-29776
        • Bridges A.
        • Gruenke L.
        • Chang Y.T.
        • Vakser I.A.
        • Loew G.
        • Waskell L.
        Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.
        J. Biol. Chem. 1998; 273: 17036-17049
        • Halpert J.R.
        Structure and function of cytochromes P450 2B: from mechanism-based inactivators to x-ray crystal structures and back.
        Drug Metab. Dispos. 2011; 39: 1113-1121
        • Scott E.E.
        • He Y.A.
        • Wester M.R.
        • White M.A.
        • Chin C.C.
        • Halpert J.R.
        • Johnson E.F.
        • Stout C.D.
        An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13196-13201
        • Banci L.
        • Bertini I.
        • Rosato A.
        • Scacchieri S.
        Solution structure of oxidized microsomal rabbit cytochrome b5. Factors determining the heterogeneous binding of the heme.
        Eur. J. Biochem. 2000; 267: 755-766
        • Nunez M.
        • Guittet E.
        • Pompon D.
        • van Heijenoort C.
        • Truan G.
        NMR structure note: oxidized microsomal human cytochrome b5.
        J. Biomol. NMR. 2010; 47: 289-295
        • Başaran N.
        • Doebler R.W.
        • Goldston H.
        • Holloway P.W.
        Effect of lipid unsaturation on the binding of native and a mutant form of cytochrome b5 to membranes.
        Biochemistry. 1999; 38: 15245-15252
        • Greenhut S.F.
        • Taylor K.M.
        • Roseman M.A.
        Tight insertion of cytochrome b5 into large unilamellar vesicles.
        Biochim. Biophys. Acta. 1993; 1149: 1-9
        • Chester D.W.
        • Skita V.
        • Young H.S.
        • Mavromoustakos T.
        • Strittmatter P.
        Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction.
        Biophys. J. 1992; 61: 1224-1243
        • Clarke T.A.
        • Im S.C.
        • Bidwai A.
        • Waskell L.
        The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.
        J. Biol. Chem. 2004; 279: 36809-36818
        • Chudaev M.V.
        • Gilep A.A.
        • Usanov S.A.
        Site-directed mutagenesis of cytochrome b5 for studies of its interaction with cytochrome P450.
        Biochemistry. 2001; 66: 667-681
        • Vergéres G.
        • Waskell L.
        Cytochrome b5, its functions, structure, and membrane topology.
        Biochimie. 1995; 77: 604-620
        • de Vries S.J.
        • van Dijk A.D.
        • Krzeminski M.
        • van Dijk M.
        • Thureau A.
        • Hsu V.
        • Wassenaar T.
        • Bonvin A.M.
        HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets.
        Proteins. 2007; 69: 726-733
        • Saribas A.S.
        • Gruenke L.
        • Waskell L.
        Overexpression and purification of the membrane-bound cytochrome P450 2B4.
        Protein Expr. Purif. 2001; 21: 303-309
        • Mulrooney S.B.
        • Waskell L.
        High level expression in Escherichia coli and purification of the membrane-bound form of cytochrome b5.
        Protein Expr. Purif. 2000; 19: 173-178
        • Dürr U.H.
        • Yamamoto K.
        • Im S.C.
        • Waskell L.
        • Ramamoorthy A.
        Solid-state NMR reveals structural and dynamical properties of a membrane-anchored electron-carrier protein, cytochrome b5.
        J. Am. Chem. Soc. 2007; 129: 6670-6671
        • Omura T.
        • Sato R.
        Isolation of cytochromes P-450 and P-420.
        Methods Enzymol. 1967; 10: 556-561
        • Harris R.K.
        • Becker E.D.
        • Cabral de Menezes S.M.
        • Goodfellow R.
        • Granger P.
        NMR nomenclature: Nuclear spin properties and conventions for chemical shifts. IUPAC recommendations 2001.
        Solid State Nucl. Magn. Reson. 2002; 22: 458-483
        • Delaglio F.
        • Grzesiek S.
        • Vuister G.W.
        • Zhu G.
        • Pfeifer J.
        • Bax A.
        NMRPipe: a multidimensional spectral processing system based on UNIX pipes.
        J. Biomol. NMR. 1995; 6: 277-293
        • Kneller D.G.
        • Kuntz I.D.
        UCSF Sparky-an NMR display, annotation, and assignment tool.
        J. Cell. Biochem. 1993; 53: 254
        • Cornilescu G.
        • Delaglio F.
        • Bax A.
        Protein backbone angle restraints from searching a database for chemical shift and sequence homology.
        J. Biomol. NMR. 1999; 13: 289-302
        • Güntert P.
        • Mumenthaler C.
        • Wüthrich K.
        Torsion angle dynamics for NMR structure calculation with the new program DYANA.
        J. Mol. Biol. 1997; 273: 283-298
        • Güntert P.
        Automated NMR structure calculation with CYANA.
        Methods Mol. Biol. 2004; 278: 353-378
        • Dvinskikh S.V.
        • Yamamoto K.
        • Ramamoorthy A.
        Heteronuclear isotropic mixing separated local field NMR spectroscopy.
        J. Chem. Phys. 2006; 125: 34507
        • Caravatti P.
        • Braunschweiler L.
        • Ernst R.R.
        Heteronuclear correlation spectroscopy in rotating solids.
        Chem. Phys. Lett. 1983; 100: 305-310
        • Fung B.M.
        • Khitrin A.K.
        • Ermolaev K.
        An improved broadband decoupling sequence for liquid crystals and solids.
        J. Magn. Reson. 2000; 142: 97-101
        • Dominguez C.
        • Boelens R.
        • Bonvin A.M.
        HADDOCK: a protein-protein docking approach based on biochemical or biophysical information.
        J. Am. Chem. Soc. 2003; 125: 1731-1737
        • Scott E.E.
        • White M.A.
        • He Y.A.
        • Johnson E.F.
        • Stout C.D.
        • Halpert J.R.
        Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-Å resolution: insight into the range of P450 conformations and the coordination of redox partner binding.
        J. Biol. Chem. 2004; 279: 27294-27301
        • Schüttelkopf A.W.
        • van Aalten D.M.
        PRODRG: a tool for high throughput crystallography of protein-ligand complexes.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1355-1363
        • Krissinel E.
        • Henrick K.
        Inference of macromolecular assemblies from crystalline state.
        J. Mol. Biol. 2007; 372: 774-797
        • Potterton E.
        • Briggs P.
        • Turkenburg M.
        • Dodson E.
        A graphical user interface to the CCP4 program suite.
        Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1131-1137
        • Zhang Q.
        • Cao C.
        • Wang Z.Q.
        • Wang Y.H.
        • Wu H.
        • Huang Z.X.
        The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H.
        Protein Sci. 2004; 13: 2161-2169
        • Lee K.B.
        • La Mar G.N.
        • Kehres L.A.
        • Fujinari E.M.
        • Smith K.M.
        • Pochapsky T.C.
        • Sligar S.G.
        1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.
        Biochemistry. 1990; 29: 9623-9631
        • Nguyen K.T.
        • Soong R.
        • Lm S.C.
        • Waskell L.
        • Ramamoorthy A.
        • Chen Z.
        Probing the spontaneous membrane insertion of a tail-anchored membrane protein by sum frequency generation spectroscopy.
        J. Am. Chem. Soc. 2010; 132: 15112-15115
        • Sanders C.R.
        • Hare B.J.
        • Howard K.P.
        • Prestegard J.H.
        Magnetically oriented phospholipid micelles as a tool for the study of membrane-associated molecules.
        Prog. Nucl. Magn. Reson. Spectrosc. 1994; 26: 421-444
        • Dürr U.H.
        • Gildenberg M.
        • Ramamoorthy A.
        The magic of bicelles lights up membrane protein structure.
        Chem. Rev. 2012; 112: 6054-6074
        • Soong R.
        • Smith P.E.
        • Xu J.
        • Yamamoto K.
        • Im S.C.
        • Waskell L.
        • Ramamoorthy A.
        Proton-evolved local-field solid-state NMR studies of cytochrome b5 embedded in bicelles, revealing both structural and dynamical information.
        J. Am. Chem. Soc. 2010; 132: 5779-5788
        • Dailey H.A.
        • Strittmatter P.
        Structural and functional properties of membrane binding segment of cytochrome b5.
        J. Biol. Chem. 1978; 253: 8203-8209
        • Holloway P.W.
        • Buchheit C.
        Topography of the membrane-binding domain of cytochrome-B5 in lipids by Fourier-transform infrared-spectroscopy.
        Biochemistry. 1990; 29: 9631-9637
        • Denny J.K.
        • Wang J.
        • Cross T.A.
        • Quine J.R.
        PISEMA powder patterns and PISA wheels.
        J. Magn. Reson. 2001; 152: 217-226
        • Nevzorov A.A.
        • Opella S.J.
        Structural fitting of PISEMA spectra of aligned proteins.
        J. Magn. Reson. 2003; 160: 33-39
        • Vergères G.
        • Waskell L.
        Expression of cytochrome b5 in yeast and characterization of mutants of the membrane-anchoring domain.
        J. Biol. Chem. 1992; 267: 12583-12591
        • Prudêncio M.
        • Ubbink M.
        Transient complexes of redox proteins: structural and dynamic details from NMR studies.
        J. Mol. Recognit. 2004; 17: 524-539
        • Zuiderweg E.R.
        Mapping protein-protein interactions in solution by NMR spectroscopy.
        Biochemistry. 2002; 41: 1-7
        • Estrada D.F.
        • Laurence J.S.
        • Scott E.E.
        Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.
        J. Biol. Chem. 2013; 288: 17008-17018
        • Volkov A.N.
        • Ferrari D.
        • Worrall J.A.
        • Bonvin A.M.
        • Ubbink M.
        The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK.
        Protein Sci. 2005; 14: 799-811
        • Tang C.
        • Iwahara J.
        • Clore G.M.
        Visualization of transient encounter complexes in protein-protein association.
        Nature. 2006; 444: 383-386
        • Volkov A.N.
        • Ubbink M.
        • van Nuland N.A.
        Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy.
        J. Biomol. NMR. 2010; 48: 225-236
        • Suh J.Y.
        • Tang C.
        • Clore G.M.
        Role of electrostatic interactions in transient encounter complexes in protein-protein association investigated by paramagnetic relaxation enhancement.
        J. Am. Chem. Soc. 2007; 129: 12954-12955
        • Matsuo H.
        • Walters K.J.
        • Teruya K.
        • Tanaka T.
        • Gassner G.T.
        • Lippard S.J.
        • Kyogoku Y.
        • Wagner G.
        Identification by NMR spectroscopy of residues at contact surfaces in large, slowly exchanging macromolecular complexes.
        J. Am. Chem. Soc. 1999; 121: 9903-9904
        • Zamoon J.
        • Nitu F.
        • Karim C.
        • Thomas D.D.
        • Veglia G.
        Mapping the interaction surface of a membrane protein: unveiling the conformational switch of phospholamban in calcium pump regulation.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4747-4752
        • Koberova M.
        • Jecmen T.
        • Sulc M.
        • Cerna V.
        • Kizek R.
        • Stiborova M.
        • Hodek P.
        Photo-cytochrome b5–a new tool to study the cytochrome P450 electron-transport chain.
        Int. J. Electrochem. Sci. 2013; 8: 125-134
        • Tamburini P.P.
        • Gibson G.G.
        Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein.
        J. Biol. Chem. 1983; 258: 13444-13452
        • Frisch C.
        • Schreiber G.
        • Johnson C.M.
        • Fersht A.R.
        Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation.
        J. Mol. Biol. 1997; 267: 696-706
        • Harel M.
        • Cohen M.
        • Schreiber G.
        On the dynamic nature of the transition state for protein-protein association as determined by double-mutant cycle analysis and simulation.
        J. Mol. Biol. 2007; 371: 180-196
        • Page C.C.
        • Moser C.C.
        • Dutton P.L.
        Mechanism for electron transfer within and between proteins.
        Curr. Opin. Chem. Biol. 2003; 7: 551-556
        • Ubbink M.
        The courtship of proteins: Understanding the encounter complex.
        FEBS Lett. 2009; 583: 1060-1066
        • Clackson T.
        • Wells J.A.
        A hot spot of binding energy in a hormone-receptor interface.
        Science. 1995; 267: 383-386
        • Sulc M.
        • Jecmen T.
        • Snajdrova R.
        • Novak P.
        • Martinek V.
        • Hodek P.
        • Stiborova M.
        • Hudecek J.
        Mapping of interaction between cytochrome P450 2B4 and cytochrome b5: the first evidence of two mutual orientations.
        Neuro Endocrinol. Lett. 2012; 33: 41-47
        • Shao W.
        • Im S.C.
        • Zuiderweg E.R.
        • Waskell L.
        Mapping the binding interface of the cytochrome b5-cytochrome c complex by nuclear magnetic resonance.
        Biochemistry. 2003; 42: 14774-14784
        • Naffin-Olivos J.L.
        • Auchus R.J.
        Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17.
        Biochemistry. 2006; 45: 755-762
        • Tamburini P.P.
        • Schenkman J.B.
        Purification to homogeneity and enzymological characterization of a functional covalent complex composed of cytochromes P-450 isozyme 2 and b5 from rabbit liver.
        Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 11-15
        • Kurnikov I.V.
        HARLEM Molecular Modeling Package. Department of Chemistry, University of Pittsburgh, Pittsburgh2000 (Version 1.0)
        • Nakamura K.
        • Horiuchi T.
        • Yasukochi T.
        • Sekimizu K.
        • Hara T.
        • Sagara Y.
        Significant contribution of arginine 112 and its positive charge of Pseudomonas putida cytochrome P-450cam in the electron transport from putidaredoxin.
        Biochim. Biophys. Acta. 1994; 1207: 40-48
        • Schlingmann K.P.
        • Kaufmann M.
        • Weber S.
        • Irwin A.
        • Goos C.
        • John U.
        • Misselwitz J.
        • Klaus G.
        • Kuwertz-Bröking E.
        • Fehrenbach H.
        • Wingen A.M.
        • Güran T.
        • Hoenderop J.G.
        • Bindels R.J.
        • Prosser D.E.
        • Jones G.
        • Konrad M.
        Mutations in CYP24A1 and idiopathic infantile hypercalcemia.
        N. Engl. J. Med. 2011; 365: 410-421
        • Rodgers K.K.
        • Pochapsky T.C.
        • Sligar S.G.
        Probing the mechanisms of macromolecular recognition-the cytochrome-b5-cytochrome c complex.
        Science. 1988; 240: 1657-1659
        • Deep S.
        • Im S.C.
        • Zuiderweg E.R.
        • Waskell L.
        Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data.
        Biochemistry. 2005; 44: 10654-10668
        • Tamburini P.P.
        • Schenkman J.B.
        Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups.
        Arch. Biochem. Biophys. 1986; 245: 512-522
        • Popovych N.
        • Sun S.
        • Ebright R.H.
        • Kalodimos C.G.
        Dynamically driven protein allostery.
        Nat. Struct. Mol. Biol. 2006; 13: 831-838
        • Ahmad S.
        • Gromiha M.
        • Fawareh H.
        • Sarai A.
        ASAView: database and tool for solvent accessibility representation in proteins.
        BMC Bioinformatics. 2004; 5: 51