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Structure and Metal Loading of a Soluble Periplasm Cuproprotein*

Open AccessPublished:August 10, 2010DOI:https://doi.org/10.1074/jbc.M110.153080
      A copper-trafficking pathway was found to enable Cu2+ occupancy of a soluble periplasm protein, CucA, even when competing Zn2+ is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu2+, but not Zn2+, quenches the fluorescence of Trp165, which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn2+ following exposure to equimolar Zn2+ and Cu2+. Cu2+-CucA is more thermodynamically stable than Zn2+-CucA but k(Zn→Cu)exchange is slow, raising questions about how the periplasm contains solely the Cu2+ form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu2+-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, ΔctaA cells overaccumulate low Mr copper complexes in the periplasm, and purified apoCucA can readily acquire Cu2+ from ΔctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.

      Introduction

      Metals diffuse through porins in the outer membrane of Gram-negative bacteria such that the periplasm is exposed to metals from the external medium (
      • Nies D.H.
      ,
      • Nikaido H.
      • Vaara M.
      ). This raises questions about how the correct metals are acquired by proteins such as CucA, which are exported in an unfolded state by the general secretory pathway and obtain metal after membrane translocation. We previously identified the most abundant Cu2+ and Mn2+ proteins, CucA and MncA, respectively, in the periplasm of the cyanobacterium Synechocystis PCC 6803 (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). MncA has Mn2+-dependent oxalate decarboxylase activity, and CucA shows some Cu2+-dependent quercetin dioxygenase activity. Folding of MncA in an extract of the soluble components of the periplasm aberrantly generates Zn2+-MncA, but MncA is a Tat substrate, which folds prior to export (
      • Santini C.L.
      • Ize B.
      • Chanal A.
      • Müller M.
      • Giordano G.
      • Wu L.F.
      ,
      • Palmer T.
      • Berks B.C.
      ,
      • Keegstra K.
      • Cline K.
      ,
      • Robinson C.
      • Bolhuis A.
      ), and so MncA can acquire Mn2+ in the cytoplasm but not the periplasm. Electron paramagnetic resonance spectra of Cu2+-MncA and Cu2+-CucA are closely similar, consistent with similarly determined and deduced metal-binding sites, respectively (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). Because CucA is exported by the general secretory pathway, if its metal-binding site is identical to MncA, then CucA might be predicted to erroneously acquire Zn2+ upon folding in the periplasm. Here, we confirm the prediction that CucA will readily bind Zn2+ and would be liable to aberrantly acquire Zn2+ rather than Cu2+ from the exchangeable metal pools in the periplasm.
      It has been proposed that copper was recruited during evolution after the appearance of atmospheric dioxygen and then exploited in the development of multicellular eukaryotes (
      • Fraústo da Silva J.J.
      • Williams R.J.
      ). It is unclear whether or not copper is needed in the cytosol of most bacteria. Moreover, few if any copper importers have been identified in bacteria. In vivo evidence that some P-type ATPases import copper (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ,
      • Odermatt A.
      • Krapf R.
      • Solioz M.
      ,
      • Lewinson O.
      • Lee A.T.
      • Rees D.C.
      ,
      • Phung L.T.
      • Ajlani G.
      • Haselkorn R.
      ,
      • Koch H.G.
      • Winterstein C.
      • Saribas A.S.
      • Alben J.O.
      • Daldal F.
      ,
      • Hassani B.K.
      • Astier C.
      • Nitschke W.
      • Ouchane S.
      ) has not yet been substantiated with biochemical data in vitro. In eukaryotes, copper proteins are extracellular or located within cytoplasmic compartments such as the trans-Golgi, vesicles derived from this network, mitochondria, or chloroplasts (
      • Kaplan J.H.
      • Lutsenko S.
      ,
      • O'Halloran T.V.
      • Culotta V.C.
      ,
      • Petris M.J.
      • Mercer J.F.
      • Culvenor J.G.
      • Lockhart P.
      • Gleeson P.A.
      • Camakaris J.
      ,
      • Lin S.J.
      • Pufahl R.A.
      • Dancis A.
      • O'Halloran T.V.
      • Culotta V.C.
      ,
      • Robinson N.J.
      • Winge D.R.
      ,
      • Setty S.R.
      • Tenza D.
      • Sviderskaya E.V.
      • Bennett D.C.
      • Raposo G.
      • Marks M.S.
      ). Copper-zinc superoxide dismutase is a rare example of a copper protein found within the eukaryotic cytosol, and it has a copper-delivery protein CCS (
      • Culotta V.C.
      • Klomp L.W.
      • Strain J.
      • Casareno R.L.
      • Krems B.
      • Gitlin J.D.
      ,
      • Changela A.
      • Chen K.
      • Xue Y.
      • Holschen J.
      • Outten C.E.
      • O'Halloran T.V.
      • Mondragón A.
      ). It is hypothesized that trafficking copper to its destinations by ligand exchange via copper metallochaperones (
      • Banci L.
      • Bertini I.
      • Ciofi-Baffoni S.
      • Kozyreva T.
      • Zovo K.
      • Palumaa P.
      ,
      • Furukawa Y.
      • Torres A.S.
      • O'Halloran T.V.
      ), coupled with compartmentalization, minimizes deleterious side reactions in the cytosol (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). A zeptomolar copper affinity of the copper sensor CueR implies that copper is either excluded or tightly bound and buffered within the Escherichia coli cytoplasm (
      • Rae T.D.
      • Schmidt P.J.
      • Pufahl R.A.
      • Culotta V.C.
      • O'Halloran T.V.
      ). In bacteria, copper-zinc superoxide dismutase is always extracellular or located within the periplasm, whereas other bacterial copper proteins are membranous, periplasmic, or extracellular. One known cytoplasmic bacterial copper enzyme is particulate methane monooxygenase (
      • Balasubramanian R.
      • Smith S.M.
      • Rawat S.
      • Yatsunyk L.A.
      • Stemmler T.L.
      • Rosenzweig A.C.
      ); however, particulate methane monooxygenase is housed in unusual internal membranes (
      • Brantner C.A.
      • Buchholz L.A.
      • Remsen C.C.
      • Collins M.L.
      ). Bacteria appear to have avoided the challenges associated with handling copper in the cytoplasm by excluding copper proteins. However, here we discover that the periplasmic copper protein CucA does not obtain its metal from periplasmic pools but requires that copper be first trafficked through the cytoplasm.
      The first crystal structures of CucA now confirm a monocupin fold and the coordination of Cu2+ via a His3 Glu1 ligand set, analogous to the Mn2+-binding residues of MncA. Trp165 is proximal to the metal-binding site, and its fluorescence is quenched by Cu2+ but not by Zn2+, allowing metal binding to be followed spectrally. In vitro metallation studies reveal ∼50% initial occupancy with Zn2+ after exposure of apoCucA to an equimolar mixture of Cu2+ and Zn2+, followed by a slow (>24 h to equilibrium) exchange to 100% Cu2+-CucA. CucA was previously identified via the fractionation of periplasm extracts using two-dimensional native liquid chromatography followed by metal analysis using inductively coupled plasma mass spectrometry (ICP-MS)
      The abbreviations used are: ICP-MS
      inductively coupled plasma mass spectrometry
      ASU
      asymmetric unit
      SeMet
      selenomethionine.
      (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). By this approach, it is possible to quantify the number of atoms of copper bound to CucA per cell. Here, the same method has been used to quantify the number of atoms of copper associated with CucA in mutants deficient in genes known to encode proteins of copper homeostasis (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ,
      • Phung L.T.
      • Ajlani G.
      • Haselkorn R.
      ,
      • Tottey S.
      • Rondet S.A.
      • Borrelly G.P.
      • Robinson P.J.
      • Rich P.R.
      • Robinson N.J.
      ,
      • Kanamaru K.
      • Kashiwagi S.
      • Mizuno T.
      ). Accumulation of Cu2+-CucA in vivo requires the same three proteins PacS, CtaA, and Atx1, which supply copper to plastocyanin within internal thylakoid compartments. Low Mr Cu2+ pools increase in abundance in periplasm extracts of ΔpacS, ΔctaA, and Δatx1 mutants, in which Cu2+-CucA fails to accumulate, showing that CucA does not directly acquire Cu2+ from these periplasmic pools. The abundance of CucA protein, but not transcripts, declines in a copper-trafficking mutant. Thus, the copper pathway does not act on expression of the cucA gene but assists the metal supply to CucA itself, and moreover, the copper supply is linked to CucA accumulation in the periplasm.

      EXPERIMENTAL PROCEDURES

      General Reagents and Bacterial Cultures

      Synechocystis PCC 6803 was cultured at 28 °C under constant light in standard BG-11 medium. Mutant strains ΔctaA (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ), ΔpacS (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ), and Δatx1 (
      • Tottey S.
      • Rondet S.A.
      • Borrelly G.P.
      • Robinson P.J.
      • Rich P.R.
      • Robinson N.J.
      ) were described previously and were maintained in the presence of kanamycin (50 μg ml−1) or chloramphenicol (7.5 μg ml−1), but antibiotics were omitted from the final experimental cultures to prevent metal chelation effects.
      E. coli BL21(DE3) cells (Novagen) were cultured in Luria-Bertani (LB) medium, and B834(DE3) cells (Novagen) were cultured in M9 minimal medium containing seleno-l-methionine (Sigma). The pET29a plasmid (Novagen) was maintained by including kanamycin (50 μg ml−1) where necessary.

      CucA Production, Purification, and in Vitro Metal Binding Studies

      Cloning of the cucA gene for heterologous expression was described previously (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). Site-directed mutagenesis of Trp165 to Phe was achieved using the QuikChange method (Stratagene) with the primers 5′-CGGATAAAACATTACCCATTGTTTTTGTTTTCATGCGTAATGAAGTTGCACCGG-3′ and 5′-CCGGTGCAACTTCATTACGCATGAAAACAAAAACAATGGGTAATGTTTTATCCG-3′. Resulting colonies were screened for the loss of a BtsCI restriction site, and a successful clone was verified by sequencing. The expression, refolding, and purification of recombinant CucA protein was performed essentially as described previously (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ), except that the final size exclusion chromatography step utilized a HiLoad 26/60 Superdex 200 column (GE Healthcare). The anion exchange-concentrated refolded protein, in 50 mm Tris, pH 7.5, 500 mm NaCl, 10 mm EDTA, was resolved on the size exclusion column in 10 mm HEPES, pH 7.0, 150 mm NaCl at 0.5 ml min−1 at 4 °C, collecting 5-ml fractions. CucA-containing fractions were assayed for protein by Coomassie assay (Pierce), and CucA was quantified by UV-visible spectroscopy (Cary 4E) using the calculated ϵ280 nm = 32,890 m−1 cm−1 for wild type and 27,930 m−1 cm−1 for the W195F mutant. Protein was verified to be metal-free (designated as containing <0.05 mol eq total) by ICP-MS.
      Fluorescence spectra were collected using a Cary Eclipse spectrofluorimeter at 20 °C. The excitation wavelength was 280 nm throughout with a 5-nm slit width, and emission was monitored between 300 and 450 nm with a 5-nm slit width, a scan rate of 120 nm min−1, 1-nm data interval, and 0.5-s averaging time. All samples contained ∼5 μm CucA in 10 mm HEPES, pH 7.0, 150 mm NaCl in quartz cuvettes (Hellma). All samples were thermally equilibrated for 10 min prior to the experiment. For titrations, metals were added by pipette from concentrated aqueous stock solutions of CuSO4, ZnSO4, or MnCl2, mixed vigorously, and then incubated at 20 °C for 60 s before the spectra were collected. All metal stocks were calibrated by ICP-MS before use. For kinetics experiments, metal was added directly to the cuvette within the spectrometer and rapidly mixed by pipetting. Emission at 320 nm was measured at 1 Hz for the duration of the experiment.

      Determination of CucA Crystal Structures

      Purified recombinant CucA (apo or metallated) was concentrated to ∼10 mg ml−1 and buffer-exchanged into 10 mm HEPES, pH 7.0, 50 mm NaCl using a YM-10 centrifugal concentrator (Centricon, Amicon). Initial crystallization trials were set up as sitting drops using a Mosquito robot (TTP Labtech) and commercially available precipitant screens. Conditions yielding crystals were scaled up and optimized in standard 24-well plates using the hanging drop method of vapor diffusion. All crystallization experiments were maintained at 20 °C. Diffraction quality crystals were produced from multiple conditions containing 0.1 m either HEPES or Tris, pH 7.5–8.8, 18–24% (w/v) PEG 8000, at a protein to precipitant solution ratio of 2 μl of protein solution to 1 μl of precipitant solution. For metallated protein, 0.5 mm CuSO4 or 0.5 mm ZnSO4 was included in the precipitant solution or 1 mm EDTA for apoprotein. Prior to data collection, crystals were flash-frozen in liquid nitrogen using the precipitant solution supplemented with 25% glycerol as a cryoprotectant. Data collection statistics and beamlines are listed in supplemental Table 1.
      Single wavelength anomalous dispersion was used to solve the crystal structure of the zinc-containing form of SeMet- substituted recombinant CucA to 2.2 Å; crystals of Cu2+-SeMet-CucA were obtained but were much smaller and did not diffract as far. Although sufficient anomalous data were collected from the copper form to permit structure solution (data not presented), the zinc structure was used as a molecular replacement model for the other CucA structures, including Cu2+-SeMet-CucA. The structure of Zn2+-CucA-SeMet was solved using the SHELX C/D/E programs as part of CCP4i. The resulting phases were used as starting phases in RESOLVE, which built the majority of the protein. The model building was completed manually in COOT with refinement carried out using Refmac5. Molecular replacement was subsequently carried out using MOLREP. Models were validated using the MOLPROBITY webserver. Structures were aligned for comparisons using LSQMAN or THESEUS. The majority of structures were obtained in the closed form; however, particularly the apoprotein or Cu2+-CucA crystallized in the absence of excess metal crystallized readily in the open form. No correlation was observed between crystal forms and the pH of the precipitant solution. The presence of copper or zinc was confirmed by calculation of anomalous difference maps from data collected at 1.378 or 1.282 Å, respectively, and by the collection of energy scans across the copper/zinc region at ESRF beamline ID29-1.

      Analysis of Copper Complexes in Periplasmic Extracts

      Isolation of periplasmic extracts from Synechocystis PCC 6803, two-dimensional liquid chromatography, ICP-MS metal analysis, SDS-PAGE, and MALDI-TOF-MS peptide mass fingerprinting used methods described previously (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ), except that HEPES buffer was used throughout in place of Tris buffer used previously.

      Isolation of RNA and Reverse Transcriptase-PCR

      Total RNA was isolated from logarithmically growing wild-type, ΔctaA, ΔpacS, and Δatx1 cells using an established method (
      • Huckle J.W.
      • Morby A.P.
      • Turner J.S.
      • Robinson N.J.
      ). RNA was treated with DNase I (Sigma), and then 1 μg of DNase-treated RNA was used for the production of cDNA using an Im-PromII reverse transcription kit (Promega). Reverse transcriptase was omitted from reaction mixtures that were used as negative controls in the RT-PCR analysis. For the PCR, specific primers for petE (forward 5′-TGCCGCTGCCAATGCAACAG-3′ and reverse 5′-GTTCACAGTAGTAGGTGTAG-3′), petJ (forward 5′-CTGTGCCGCTTGTCATAATG-3′ and reverse 5′-AGCACGTAGGCCGCCACATC-3′), cucA (forward 5′-TATTTTCAAGCTGTGGGGC-3′ and reverse 5′-CGGTTAAAAGCTTCAATTAC-3′), and rps1 (forward 5′-CTCTGATTGACATTGGGGCG-3′ and reverse 5′-GAGCGCTGATGTGGGAGCCG-3′) were used, which were designed to amplify an ∼300-bp region internal to each gene. Cycling conditions, using Taq polymerase (New England Biolabs), included an initial denaturation step at 95 °C for 2 min and then 25–30 cycles of denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 20 s, followed by a final extension step at 72 °C for 5 min. Products were analyzed on 1% (w/v) agarose gels stained with ethidium bromide.

      Metal Association with CucA Using Periplasm Extracts

      Periplasm extracts were prepared from 1 to 2 liters of wild-type or ΔctaA Synechocystis PCC 6803 culture, into a final volume of 100 ml of water, as described previously (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). This extract was divided equally into two portions, to which was added 1 ml of 10 mm HEPES, pH 7.0, 150 mm NaCl buffer (denoted “control”) or 1 ml of 1.5 μm CucA in the same buffer (denoted “CucA”). The samples were mixed and incubated for 1 h at 4 °C, and then 1 m HEPES, pH 8.8, was added to each sample to a final concentration of 20 mm. Each sample was independently loaded onto a 1-ml HiTrap Q HP anion exchange column (GE Healthcare), washed in 20 ml of 20 mm HEPES, pH 8.8, and then eluted in 1 ml of 20 mm HEPES, pH 8.8, 400 mm NaCl. Aliquots (200 μl) of each eluant fraction were resolved by HPLC size exclusion chromatography (TSK SW3000, Tosoh Biosciences) in 5 mm HEPES, pH 7.0, 50 mm NaCl at 0.5 ml min−1 collecting 0.5-ml fractions. Each fraction was analyzed for metal by ICP-MS.

      RESULTS

      Visualization of the CucA Metal Site Adjacent to Trp165

      The structure of CucA was solved, initially for the purpose of visualizing its metal site to identify the metal ligands and to establish whether or not it is a cupin, as predicted through bioinformatics. Diffraction quality crystals of Cu2+-SeMetCucA (four molecules per asymmetric unit (ASU)), Cu2+-CucA (four and one molecule per ASU), Zn2+-SeMetCucA (two molecules per ASU), and apoSeMetCucA (four molecules per ASU) were analyzed (supplemental Table 1). The Sec-dependent signal sequence of CucA is missing from the recombinant protein, and in addition, four amino-terminal residues were not detected in the electron density maps so that the structures commence at residue 35. CucA is confirmed to exhibit a monocupin fold with the metal-binding site positioned within a relatively hydrophobic cavity (Fig. 1a). In metal-bound forms, Cu2+ or Zn2+, the metal ion is coordinated to the same four residues, three histidines, His88,90,149, plus Glu95 (Fig. 1b), identical to the protein metal-binding ligands of Mn2+-MncA (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). Bonds to Cu2+ and Zn2+ are ∼2.1 Å (respective metal identities confirmed at edge wavelengths of 1.378 and 1.282 Å) (Fig. 1, c and d). Trp165 is proximal to the metal at 4.4 Å (Fig. 1b), and therefore, its fluorescence is liable to be quenched through Förster dipole coupling of the Trp excited state with metal-derived charge transfer transitions of suitable energies (
      • Whittaker M.M.
      • Mizuno K.
      • Bächinger H.P.
      • Whittaker J.W.
      ).
      Figure thumbnail gr1
      FIGURE 1Crystal structure of Cu2+-CucA. a, structural elements (α, α-helices; β, β-strands; l, loops), colored in order from the amino (blue) to carboxyl (red) terminus, are numerically labeled. b, residues (green) surrounding the copper atom (blue sphere) of Cu2+-CucA are shown in the closed conformation. c, residues surrounding the metal atoms within CucA with corresponding composite omit electron density maps each contoured at 1.5 σ for Cu2+-CucA, and d, Zn2+-CucA.
      The metal binding geometry of both Cu2+-CucA and Zn2+-CucA to protein ligands approximates to distorted tetrahedral arrangement, but one or two water molecules provide additional fifth and possibly sixth ligands at variable distances. One water molecule is at a comparable distance to the other ligands (1.9–2.4 Å), and the second tends to be longer, perhaps not coordinating at all (2.4–3.2 Å), generating a distorted square pyramidal arrangement. Cu(II) has a preference for square planar or square pyramidal Zn(II) for tetrahedral geometry, and hence CucA imposes a geometry that may influence its metal-binding preferences. The surrounding protein structure is comparable between the two metal forms (Cu2+ versus Zn2+). More than one crystal conformer of CucA was observed (supplemental Fig. 1), with some analogy to the open and closed forms of oxalate decarboxylase (
      • Anand R.
      • Dorrestein P.C.
      • Kinsland C.
      • Begley T.P.
      • Ealick S.E.
      ,
      • Just V.J.
      • Stevenson C.E.
      • Bowater L.
      • Tanner A.
      • Lawson D.M.
      • Bornemann S.
      ). Both open and closed conformations of Cu2+-CucA have been detected. The open forms contain a channel to the metal-binding site that is expected to allow substrate entry and product release. Although the endogenous substrates of Cu2+-CucA are unknown, the enzyme exhibits some quercetin dioxygenase activity that is absent in Zn2+-CucA (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). However, the channel to the CucA metal site and the presumed substrate cavity is small compared with known quercetin dioxygenases (
      • Steiner R.A.
      • Kalk K.H.
      • Dijkstra B.W.
      ), suggesting that the in vivo substrate is likely to be smaller than quercetin. The entry pocket is lined by Glu58, Phe60, Pro85, and Phe209 with a single string of water molecules connecting the metal site to the surface (supplemental Fig. 2). In the alternative form of CucA, the channel is occluded by the movement of residues 52–60, resulting in Glu58 and Ala56 and the loop 1 region projecting into the channel (supplemental Fig. 3). The B-factors are higher in open CucA, notably in the regions that close the channel in the alternative form (supplemental Fig. 4), and several residues (203–207 inclusive) from helix α2 and loop 11 could not be modeled in this open form.

      Binding of Cu2+ and Zn2+ to CucA in Vitro

      In vitro metal-binding experiments previously suggested that MncA might prefer Zn2+ relative to Cu2+, with approximately 1 order of magnitude difference in stability constants (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). Because the CucA ligands are identical to those of MncA (Fig. 1b) its preference for Cu2+ relative to Zn2+ was therefore tested (Fig. 2). Intrinsic fluorescence of CucA is quenched by 1 eq of Cu2+ (Fig. 2a) but not Zn2+ (Fig. 2b), although CucA does bind 1 eq of Zn2+ (Fig. 2e). Cu2+-dependent quenching was lost when Trp165 was substituted with Phe, and this CucA variant had a low level of fluorescence similar to that of wild-type CucA saturated with Cu2+ (Fig. 2c). These data support Trp165 as the source of fluorescence that is quenched by Cu2+. Unlike Zn2+, which is spectrally silent due to its d10 electron configuration, charge transfer transitions are common with Cu2+. Quenching of fluorescence by Cu2+, but not Zn2+, is thus consistent with a Förster dipole coupling mechanism.
      Figure thumbnail gr2
      FIGURE 2Copper and zinc both bind to CucA. a, quenching of the fluorescence emission spectra of CucA (5 μm in 10 mm HEPES, pH 7.0, 150 mm NaCl) exposed to increasing concentrations of Cu2+, 280 nm excitation. b, lack of quenching of the fluorescence emission spectra of CucA, as in a, exposed to increasing concentrations of Zn2+. c, fluorescence emission of CucA at 320 nm as a function of added Zn2+ (diamonds), Cu2+ (triangles), and simultaneous addition of equimolar Zn2+ plus Cu2+ to 5 μm apoCucA (circles). Fluorescence was measured 60 s after metal addition and mixing. Fluorescence emission of a CucA W165F variant was also determined (closed squares). Fractionation of CucA-bound metal by size exclusion chromatography with metal (open diamonds, zinc; open triangles, copper) detected by ICP-MS after addition of 10 μm Cu2+ (d), 10 μm Zn2+ (e), and 10 μm Zn2+ plus 10 μm Cu2+ (f) to 5 μm apoCucA (closed circles, protein).
      Metallation of apoCucA with equimolar Zn2+ and Cu2+ initially causes only partial quenching of fluorescence (Fig. 2c). The initial generation of a mixture of Zn2+-CucA and Cu2+-CucA was confirmed by analyzing bound metal by ICP-MS after fractionation by size exclusion chromatography (Fig. 2f). Prolonged incubation for 24 h leads to further quenching approaching that observed following the addition of Cu2+ alone (Fig. 3a). This implies slow Zn2+ to Cu2+ exchange, with Cu2+ ultimately forming the more thermodynamically stable complex at equilibrium. The rate of replacement of Zn2+ by Cu2+ is concentration-dependent (Fig. 3b). EDTA (1 mm) out-competes apoCucA (5 μm) for Cu2+ (Fig. 4a), and a dissociation rate for Cu2+-CucA of kCu,OFF = 7.5 × 10−7 s−1 was thus estimated from the rate of increase in Cu2+-CucA fluorescence in the presence of a similar excess of EDTA (Fig. 4b). The initial formation of an approximately equal mixture of Zn2+-CucA and Cu2+-CucA (Fig. 2f) implies that the two metals have similar association rates, and therefore a dissociation rate for Zn2+-CucA of kZn,OFF = 4.6 × 10−5 s−1 was estimated from the rate of metal exchange in the presence of surplus Cu2+ (Fig. 3b). A similar value of kZn,OFF = 5.0 × 10−5 s−1 was also estimated by detecting loss of Zn2+ from CucA by ICP-MS after 48 h (data not shown).
      Figure thumbnail gr3
      FIGURE 3Zn2+-CucA exchanges slowly with Cu2+. a, quenching of the fluorescence emission of apoCucA (5 μm) as a function of time following simultaneous addition of equimolar Zn2+ and Cu2+ (both at 2.5 m excess with respect to CucA). b, quenching of the fluorescence emission of Zn2+-CucA (2.5 m excess relative to CucA) as a function of time following addition of Cu2+ at increasing molar eq relative to Zn2+ (1:1, 2:1, 5:1, 10:1, and 20:1).
      Figure thumbnail gr4
      FIGURE 4Dissociation of Cu2+-CucA is slow. a, intrinsic fluorescence of CucA (5 μm) is not quenched by 2.5 m excess Cu2+ added in the presence of EDTA (1 mm). b, slow restoration of intrinsic fluorescence of CucA after initial quenching with 2.5 m eq of Cu2+ followed by the addition of excess EDTA (1 mm). Inset is plotted on an expanded y axis to show the rate of Cu2+-CucA dissociation.
      Thus, the metal site of CucA prefers Cu2+ to Zn2+, but if Zn2+ binds first, the subsequent replacement with Cu2+ is slow. Under these biochemical/biophysical conditions, partial occupancy with Zn2+ might be predicted to occur in vivo at times when the periplasm does not contain a sufficient excess of Cu2+ relative to Zn2+. In natural environments periplasm Cu2+ levels are likely to be much more limiting than in nutrient-rich BG-11 culture medium that contains 0.3 μm copper. Conversely, BG-11 medium contains a surplus of zinc relative to copper, yet CucA is occupied by copper.

      Copper Transporters CtaA and PacS, Plus the Atx1 Metallochaperone, Are Required for Formation of Cu2+-CucA

      Copper is required for plastocyanin and cytochrome oxidase in this bacterium, both of which are located within internal membrane-bound compartments called thylakoids (Fig. 5a). Mutants deficient in either of two P1-type ATPases, CtaA and PacS, or a soluble metallochaperone Atx1 (Fig. 5a), show phenotypes consistent with impaired copper supply to these cytoplasmic enzymes (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ,
      • Tottey S.
      • Rondet S.A.
      • Borrelly G.P.
      • Robinson P.J.
      • Rich P.R.
      • Robinson N.J.
      ). Here, we have used native two-dimensional liquid chromatography followed by inductively coupled plasma-mass spectrometry of periplasm extracts to compare the number of atoms of Cu2+ bound to CucA per cell, in the Δatx1, ΔctaA, and ΔpacS mutants (Fig. 5, b and c). The Cu2+ pool in periplasm extracts corresponding to CucA was identified previously by mass fingerprinting after using principal component analysis to correlate Cu2+ with proteins that had been further resolved using denaturing PAGE (
      • Tottey S.
      • Waldron K.J.
      • Firbank S.J.
      • Reale B.
      • Bessant C.
      • Sato K.
      • Cheek T.R.
      • Gray J.
      • Banfield M.J.
      • Dennison C.
      • Robinson N.J.
      ). Here, we find that the loss of any of the three proteins that supply copper to plastocyanin also leads to a decline in Cu2+-CucA (Fig. 5, b and c). The phenotype is weakest for mutants lacking the copper metallochaperone Atx1, implying that its role in the pathway is partly redundant, although in the absence of either of the copper ATPases, CtaA, or PacS, negligible Cu2+ reaches CucA. This has some analogy to the loss of cbb3 oxidase in Rhodobacter capsulatus on deletion of the hypothetical copper-ATPase CcoI (
      • Koch H.G.
      • Winterstein C.
      • Saribas A.S.
      • Alben J.O.
      • Daldal F.
      ). Notably, the cbb3 oxidase is a membrane protein with an intramembrane CuB site and hence dissimilar to the periplasm localization of CucA.
      Figure thumbnail gr5
      FIGURE 5CucA obtains less Cu2+ in trafficking mutants. a, cuproproteins CucA, plastocyanin and cytochrome oxidase (CcO), are located in the periplasm and thylakoid compartments, respectively. Copper metallochaperone Atx1 is cytosolic and interacts with amino-terminal regions of copper ATPases CtaA and PacS. PacS is predominantly located in thylakoid membranes. b, profile of periplasmic copper complexes from wild-type and mutant cells, separated using two-dimensional liquid chromatography based on relative molecular mass (Mr) and charge (pI). c, number of atoms of Cu2+ cell−1 bound to CucA in wild-type and mutant cells calculated from the volumes under CucA peaks (as in b). Data are the means of three independent replicates with S.D. of triplicates.

      Expression of the cucA Gene Does Not Respond to Copper

      Loss of Cu2+-CucA in ΔctaA, ΔpacS, or Δatx1 mutant cells could either result from altered expression of the cucA gene due to loss of copper supply to a metal-sensing transcriptional regulator or impaired Cu2+ delivery to apoCucA. The plastocyanin gene petE is switched off in low copper and the cytochrome c6 gene petJ is switched on, to allow the substitution of copper in plastocyanin with heme iron in cytochrome c6 for photosynthetic electron transport (
      • Zhang L.
      • McSpadden B.
      • Pakrasi H.B.
      • Whitmarsh J.
      ,
      • Merchant S.
      • Bogorad L.
      ). These two genes were therefore used as controls in an analysis of copper-responsive gene expression. No change in the abundance of cucA transcripts was detected in any of the mutants, although a decrease in petE and an increase in petJ transcripts was most evident in cells missing either copper transporter (Fig. 6a). This implies that the two transporters and the metallochaperone are involved in Cu2+ supply to CucA itself rather than to a transcriptional regulator of the cucA gene.
      Figure thumbnail gr6
      FIGURE 6Loss of CucA protein but not transcripts in Cu2+-trafficking mutants. a, RT-PCRs using RNA isolated from the four genotypes and primers corresponding to the four transcripts as shown. All products are reverse transcriptase-dependent (). 25 cycles, plus 30 cycles for cucA, were used. Similar trends were observed in two further experiments using independent RNA isolates. b, SDS-PAGE of crude periplasm extracts of wild-type and ΔctaA mutants after concentration using ion exchange chromatography.
      The CucA protein is undetected in a crude periplasm extract from the ΔctaA mutant (Fig. 6b). There are at least two similarly migrating bands in the region of CucA on Fig. 6b, and mass fingerprinting confirms that these are not CucA in ΔctaA mutants (supplemental Table 2). Loss of CucA protein implies that some facet of the production of CucA, its localization or stability, depends upon acquisition of the correct metal that in turn depends upon a copper-trafficking pathway.

      Purified CucA Can Acquire Cu2+ from ΔctaA Periplasm Extracts in Vitro

      Additional low Mr Cu2+-complexes accumulate in the periplasm of each of the transport and trafficking mutants (Fig. 5b). Presumably these represent a source of copper that would normally be imported across the cytoplasmic membrane and trafficked to CucA via the actions of CtaA, Atx1, and PacS. The chemical nature of these low Mr periplasm copper complexes is unknown, but they might include copper-glutathione in the light of evidence of periplasm-localized glutathione (
      • Pittman M.S.
      • Robinson H.C.
      • Poole R.K.
      ). We have examined whether or not these complexes could provide a source of copper for CucA by adding purified apoCucA to periplasm extracts and determining the metal status of the recombinant protein. By using extracts from wild-type cells, CucA does acquire some zinc (0.22 mol eq assuming full recovery of CucA) (Fig. 7), supporting the concept that some mechanism will indeed be required to achieve full copper occupancy in vivo, especially after growth in natural environments with less copper availability. Most importantly, using similar extracts from ΔctaA mutants, recombinant CucA became fully populated with copper (0.95 eq assuming full recovery of CucA) (Fig. 7), suggesting that the low Mr copper complexes that accumulate in these cells are suitable copper donors yet fail to form Cu2+-CucA in ΔctaA mutants (Fig. 5). Thus, in living cells apoCucA does not directly acquire Cu2+ from the low Mr pools in the periplasm, rather these ions must be trafficked through the cytoplasm for Cu2+-CucA to accumulate in the periplasm implying that trans-membrane copper transport is coupled to protein production and/or delivery to the periplasm.
      Figure thumbnail gr7
      FIGURE 7In vitro apoCucA acquires copper from ΔctaA but zinc and copper from wild-type periplasm extracts. Purified CucA protein (1 ml of 1.5 μm in 10 mm HEPES, pH 7.0, 150 mm NaCl) (open symbols) or buffer control (closed symbols) was added to periplasm extracts, which were then concentrated by anion exchange and eluted metal complexes resolved by gel filtration HPLC (TSK SW-3000) and eluant analyzed for copper (triangles) and zinc (diamonds) by ICP-MS.

      DISCUSSION

      Ab initio structures of CucA reveal a monocupin that binds divalent metals via three histidine imidazoles and a glutamate carboxylate within a hydrophobic pocket (Fig. 1). Dissociation rates for Zn2+ and Cu2+ are slow, in the region of kZn,OFF = 4.6 × 10−5 s−1 and kCu,OFF = 7.5 × 10−7 s−1, respectively, allowing only slow replacement of Zn2+ with Cu2+ (FIGURE 2, FIGURE 3, FIGURE 4). Association rates for Zn2+ and Cu2+ are both rapid (kZn,ONkCu,ON) such that incubation of apoCucA with equimolar Zn2+ and Cu2+ generates a mixed population of Zn2+-CucA and Cu2+-CucA that only gradually (t = days) resolves to the more thermodynamically stable Cu2+ form (FIGURE 2, FIGURE 3). Cu2+ acquisition in vivo is facilitated by a copper-trafficking pathway involving two P1-type ATPases and copper metallochaperone (Fig. 5), the same proteins also supply copper to plastocyanin within thylakoids (
      • Tottey S.
      • Rich P.R.
      • Rondet S.A.
      • Robinson N.J.
      ,
      • Tottey S.
      • Rondet S.A.
      • Borrelly G.P.
      • Robinson P.J.
      • Rich P.R.
      • Robinson N.J.
      ).
      Why is CucA Cu2+ acquisition assisted by a trafficking pathway in vivo? Maintaining a pool of exchangeable Cu2+ in the periplasm, sufficient to correctly populate CucA, would be challenging in natural environments with low concentrations of Cu2+. Presumably, the trafficking pathway encourages occupancy of CucA with Cu2+ and not some other metal such as Zn2+. Notably, partial occupancy of apoCucA with Zn2+ occurs when apoCucA acquires metal from periplasm extracts of wild-type cells cultured in standard medium that is copper-rich relative to natural habitats (Fig. 7).
      CucA is missing from the periplasm of copper-trafficking mutants revealing that cofactor insertion into CucA is necessary for the protein to accumulate (Fig. 6b and supplemental Table 2). This is not a function of Cu2+-dependent expression of the cucA gene because cucA transcripts show normal levels of abundance in both ΔctaA and ΔpacS cells (Fig. 6a). It is well established that many apoproteins are more susceptible to proteolytic degradation in vivo than their metal-bound holo-forms. A reasonable hypothesis might have been that in ΔctaA, ΔpacS, and Δatx1 mutants, CucA fails to acquire sufficient Cu2+ and hence is rapidly degraded in the periplasm. However, mutants in either of the copper-transporting ATPases accumulate additional low Mr copper complexes in the periplasm (Fig. 5b). Exchangeable copper pools in extracts of the periplasm can readily donate Cu2+ to apoCucA in vitro, and this is especially true of the pools in ΔctaA cells (Fig. 7). Periplasm extracts of ΔctaA cells grown in standard (copper-replete) culture medium provide sufficient Cu2+ to saturate an aliquot of apoCucA, unlike the partial Zn2+ occupancy generated using analogous extracts from wild-type cells (Fig. 7). This strongly argues that apoCucA fails to reach the periplasm in copper-trafficking mutants because any apoCucA that had been translated and translocated in ΔctaA cells should have readily acquired copper and become refractory to proteolysis, yet the protein was not detected.
      Cyanobacteria are unlike most bacteria in possessing the internal membrane compartments, thylakoids, that house the copper proteins plastocyanin and cytochrome oxidase (Fig. 5a). There is some evidence that vesicle trafficking occurs between the plasma and thylakoid membranes (
      • Schneider D.
      • Fuhrmann E.
      • Scholz I.
      • Hess W.R.
      • Graumann P.L.
      ,
      • Nevo R.
      • Charuvi D.
      • Shimoni E.
      • Schwarz R.
      • Kaplan A.
      • Ohad I.
      • Reich Z.
      ), which might suggest that CucA could acquire copper in thylakoids before being trafficked to the periplasm. However, multiple proteomics studies have established that the product of open reading frame sll1785 (namely CucA) is uniquely localized to the periplasm, never the thylakoids; and furthermore, the Sec-dependent signal sequence of CucA has been exploited in multivariate analyses to distinguish periplasm targeting signals from those of proteins targeted to thylakoids (
      • Rajalahti T.
      • Huang F.
      • Klement M.R.
      • Pisareva T.
      • Edman M.
      • Sjöström M.
      • Wieslander A.
      • Norling B.
      ). Thus, the implication is that translation and translocation of CucA across the plasma membrane is somehow linked to outward copper transport. Future studies should address the nature of this linkage and why it requires both CtaA and PacS. In the related cyanobacterium Synechococcus PCC 7942, PacS was mainly localized to thylakoid membranes by Western blotting using an anti-PacS antiserum, with a smaller subpopulation potentially located at the plasma membranes (
      • Kanamaru K.
      • Kashiwagi S.
      • Mizuno T.
      ).
      Accumulation of low Mr Cu2+ pools in the periplasm of mutants missing components of the trafficking pathway (Fig. 5b) implies that these pools are the source of copper for the pathway, but crucially copper from these pools is routed in and out of the cytoplasm to form Cu2+-CucA in vivo. Prokaryotes, including E. coli, that are devoid of known cytosolic cuproproteins and devoid of homologues of known cytosolic copper chaperones (such as CopZ or Atx1) must nonetheless have alternative mechanisms to inhibit deleterious side reactions of copper in the cytosol if these ions must pass through this compartment to supply cuproproteins in the periplasm.

      Acknowledgments

      We thank Andrew McCarthy, ESRF, Grenoble, France, for collecting scans and data at the edges for metal validation.

      Supplementary Material

      REFERENCES

        • Nies D.H.
        Nies D.H. Silver S. Molecular Microbiology of Heavy Metals. Springer-Verlag, Heidelberg, Germany2007: 117-142
        • Nikaido H.
        • Vaara M.
        Microbiol. Rev. 1985; 49: 1-32
        • Tottey S.
        • Waldron K.J.
        • Firbank S.J.
        • Reale B.
        • Bessant C.
        • Sato K.
        • Cheek T.R.
        • Gray J.
        • Banfield M.J.
        • Dennison C.
        • Robinson N.J.
        Nature. 2008; 455: 1138-1142
        • Santini C.L.
        • Ize B.
        • Chanal A.
        • Müller M.
        • Giordano G.
        • Wu L.F.
        EMBO J. 1998; 17: 101-112
        • Palmer T.
        • Berks B.C.
        Microbiology. 2003; 149: 547-556
        • Keegstra K.
        • Cline K.
        Plant Cell. 1999; 11: 557-570
        • Robinson C.
        • Bolhuis A.
        Biochim. Biophys. Acta. 2004; 1694: 135-147
        • Fraústo da Silva J.J.
        • Williams R.J.
        The Biological Chemistry of the Elements. Oxford University Press, Oxford, UK2001
        • Tottey S.
        • Rich P.R.
        • Rondet S.A.
        • Robinson N.J.
        J. Biol. Chem. 2001; 276: 19999-20004
        • Odermatt A.
        • Krapf R.
        • Solioz M.
        Biochem. Biophys. Res. Commun. 1994; 202: 44-48
        • Lewinson O.
        • Lee A.T.
        • Rees D.C.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4677-4682
        • Phung L.T.
        • Ajlani G.
        • Haselkorn R.
        Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 9651-9654
        • Koch H.G.
        • Winterstein C.
        • Saribas A.S.
        • Alben J.O.
        • Daldal F.
        J. Mol. Biol. 2000; 297: 49-65
        • Hassani B.K.
        • Astier C.
        • Nitschke W.
        • Ouchane S.
        J. Biol. Chem. 2010; 285: 19330-19337
        • Kaplan J.H.
        • Lutsenko S.
        J. Biol. Chem. 2009; 284: 25461-25465
        • O'Halloran T.V.
        • Culotta V.C.
        J. Biol. Chem. 2000; 275: 25057-25060
        • Petris M.J.
        • Mercer J.F.
        • Culvenor J.G.
        • Lockhart P.
        • Gleeson P.A.
        • Camakaris J.
        EMBO J. 1996; 15: 6084-6095
        • Lin S.J.
        • Pufahl R.A.
        • Dancis A.
        • O'Halloran T.V.
        • Culotta V.C.
        J. Biol. Chem. 1997; 272: 9215-9520
        • Robinson N.J.
        • Winge D.R.
        Annu. Rev. Biochem. 2010; 79: 537-562
        • Setty S.R.
        • Tenza D.
        • Sviderskaya E.V.
        • Bennett D.C.
        • Raposo G.
        • Marks M.S.
        Nature. 2008; 454: 1142-1146
        • Culotta V.C.
        • Klomp L.W.
        • Strain J.
        • Casareno R.L.
        • Krems B.
        • Gitlin J.D.
        J. Biol. Chem. 1997; 272: 23469-23472
        • Changela A.
        • Chen K.
        • Xue Y.
        • Holschen J.
        • Outten C.E.
        • O'Halloran T.V.
        • Mondragón A.
        Science. 2003; 301: 1383-1387
        • Banci L.
        • Bertini I.
        • Ciofi-Baffoni S.
        • Kozyreva T.
        • Zovo K.
        • Palumaa P.
        Nature. 2010; 465: 645-648
        • Furukawa Y.
        • Torres A.S.
        • O'Halloran T.V.
        EMBO J. 2004; 23: 2872-2881
        • Rae T.D.
        • Schmidt P.J.
        • Pufahl R.A.
        • Culotta V.C.
        • O'Halloran T.V.
        Science. 1999; 284: 805-808
        • Balasubramanian R.
        • Smith S.M.
        • Rawat S.
        • Yatsunyk L.A.
        • Stemmler T.L.
        • Rosenzweig A.C.
        Nature. 2010; 465: 115-119
        • Brantner C.A.
        • Buchholz L.A.
        • Remsen C.C.
        • Collins M.L.
        Curr. Microbiol. 2000; 40: 132-134
        • Tottey S.
        • Rondet S.A.
        • Borrelly G.P.
        • Robinson P.J.
        • Rich P.R.
        • Robinson N.J.
        J. Biol. Chem. 2002; 277: 5490-5497
        • Kanamaru K.
        • Kashiwagi S.
        • Mizuno T.
        Mol. Microbiol. 1994; 13: 369-377
        • Huckle J.W.
        • Morby A.P.
        • Turner J.S.
        • Robinson N.J.
        Mol. Microbiol. 1993; 7: 177-187
        • Whittaker M.M.
        • Mizuno K.
        • Bächinger H.P.
        • Whittaker J.W.
        Biophys. J. 2006; 90: 598-607
        • Anand R.
        • Dorrestein P.C.
        • Kinsland C.
        • Begley T.P.
        • Ealick S.E.
        Biochemistry. 2002; 41: 7659-7669
        • Just V.J.
        • Stevenson C.E.
        • Bowater L.
        • Tanner A.
        • Lawson D.M.
        • Bornemann S.
        J. Biol. Chem. 2004; 279: 19867-19874
        • Steiner R.A.
        • Kalk K.H.
        • Dijkstra B.W.
        Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16625-16630
        • Zhang L.
        • McSpadden B.
        • Pakrasi H.B.
        • Whitmarsh J.
        J. Biol. Chem. 1992; 267: 19054-19059
        • Merchant S.
        • Bogorad L.
        Mol. Cell. Biol. 1986; 6: 462-469
        • Pittman M.S.
        • Robinson H.C.
        • Poole R.K.
        J. Biol. Chem. 2005; 280: 32254-32261
        • Schneider D.
        • Fuhrmann E.
        • Scholz I.
        • Hess W.R.
        • Graumann P.L.
        BMC Cell Biol. 2007; 8: 39-48
        • Nevo R.
        • Charuvi D.
        • Shimoni E.
        • Schwarz R.
        • Kaplan A.
        • Ohad I.
        • Reich Z.
        EMBO J. 2007; 26: 1467-1473
        • Rajalahti T.
        • Huang F.
        • Klement M.R.
        • Pisareva T.
        • Edman M.
        • Sjöström M.
        • Wieslander A.
        • Norling B.
        J. Proteome Res. 2007; 6: 2420-2434