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Originally published In Press as doi:10.1074/jbc.M702696200 on May 18, 2007

J. Biol. Chem., Vol. 282, Issue 30, 22185-22194, July 27, 2007
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Crystal Structures of Saccharomyces cerevisiae N-Myristoyltransferase with Bound Myristoyl-CoA and Inhibitors Reveal the Functional Roles of the N-terminal Region*

Jian Wu{ddagger}§, Yong Tao, Meilan Zhang{ddagger}, Michael H. Howard, Steven Gutteridge1, and Jianping Ding{ddagger}2

From the {ddagger}State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and the §Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China and the DuPont Stine Haskell Research Center, Newark, Delaware 19714

Received for publication, March 29, 2007 , and in revised form, May 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Protein N-myristoylation catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT) plays an important role in a variety of critical cellular processes and thus is an attractive target for development of antifungal drugs. We report here three crystal structures of Saccharomyces cerevisiae NMT: in binary complex with myristoyl-CoA (MYA) alone and in two ternary complexes involving MYA and two different non-peptidic inhibitors. In all three structures, the majority of the N-terminal region, absent in all previously reported structures, forms a well defined motif that is located in the vicinity of the peptide substrate-binding site and is involved in the binding of MYA. The Ab loop, which might be involved in substrate recognition, adopts an open conformation, whereas a loop of the N-terminal region (residues 22-24) that covers the top of the substrate-binding site is in the position occupied by the Ab loop when in the closed conformation. Structural comparisons with other NMTs, together with mutagenesis data, suggest that the N-terminal region of NMT plays an important role in the binding of both MYA and peptide substrate, but not in subsequent steps of the catalytic mechanism. The two inhibitors occupy the peptide substrate-binding site and interact with the protein through primarily hydrophobic contacts. Analyses of the inhibitorenzyme interactions provide valuable information for further improvement of antifungal inhibitors targeting NMT.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Myristoyl-CoA:protein N-myristoyltransferases (NMTs3; EC 2.3.1.97 [EC] ) are a family of enzymes belonging to the GCN5-related N-acetyltransferase superfamily (for reviews, see Refs. 1 and 2). They catalyze the covalent linkage of myristate, a rare 14-carbon saturated fatty acid (tetradecanoate, C14:0), from myristoyl-CoA (MYA) to the N-terminal glycine of proteins, a process that occurs following the removal of the initiator methionine residue of growing polypeptide chains (3, 4). This co-translational modification is an essential prelude to these altered proteins fully participating in important cellular processes, including signal transduction cascades and vesicular and protein trafficking (4-8). Typically, the consequences of attaching an extended alkyl chain to the protein result in increased lipophilicity, facilitating its association with cellular/subcellular membranes and mediating interactions with other proteinaceous partners (for reviews, see Refs. 3, 9, and 10).

NMTs have been characterized from a broad range of eukaryotic sources, including Saccharomyces cerevisiae (11), Candida albicans (12), Cryptococcus neoformans (13), Plasmodium falciparum (14), and Homo sapiens (15). Kinetic studies with MYA and peptide substrates or their analogs have shown that NMT catalysis conforms to an ordered Bi Bi reaction mechanism (16). Initial binding of MYA to the enzyme induces a conformational change and subsequent formation of a binding site for the protein substrate. Following the reaction, CoA is released prior to dissociation of the myristoylated peptide.

Genetic and biochemical data have established that NMTs are essential for growth and survival of a number of human yeast strains and parasites, such as C. albicans and C. neoformans (17) and the protozoa Leishmania major and Trypanosoma brucei (18). Sequence comparison and biochemical studies have shown that human and fungal NMTs share high sequence conservation at the MYA-binding site, but have divergent peptide substrate specificities (3). These properties make NMT an attractive therapeutic target for antifungal agents (19-21) designed to occupy the peptide substrate-binding site. In addition, because of its essential role in cell viability, NMT is a potential target for antiviral, antiparasitic, and even antineoplastic chemotherapy (18, 22-24). Thus, understanding the structural basis of the recognition and binding of the substrates with the enzyme should be useful for the development of new therapeutic agents.

Crystal structures of NMTs from two species have been determined, including those of C. albicans NMT in apo-form, in binary complexes with several non-peptidic inhibitors, and in ternary complex with MYA and the peptide inhibitor SC-58272 (25, 26), and structures of S. cerevisiae (Sc) NMT in binary complex with MYA and in ternary complex with a non-hydrolyzable MYA analog (S-(2-oxo)pentadecyl-CoA) and SC-58272 or a peptide substrate (GLYASKLA) (27, 28). Structural and biochemical data have defined how the substrates are bound relative to each other at the active site and have revealed insights into the substrate binding specificity and the catalytic mechanism. However, the N-terminal region of NMTs, which has been implicated to play a pivotal role in the subcellular localization of NMT in mammalian cells (29), is absent in all of those structures because it is either disordered in the structures or removed to facilitate the structural studies.

Kinetic studies of synthetic peptides based on the N-terminal sequences of known N-myristoylproteins indicate that optimum peptide substrates for ScNMT have an absolutely conserved Gly residue at position +1 and a marked preference for Ser and Lys at positions +5 and +6, respectively (9, 30). Based on this preference, a large number of peptide-based and peptide-like inhibitors that are effective against C. albicans and C. neoformans (19-21, 31) and that have antiparasitic activity (24) have been developed. Because of the limitations of peptide-based therapeutics, however, alternative small non-peptidic inhibitors have been screened, and some have been identified with very high activity and selectivity against NMTs of pathogenic fungal species (26, 32, 33).

We report here the crystal structures of full-length ScNMT in binary complex with MYA and in ternary complexes with MYA and Compounds I and II, two distinct non-peptidic inhibitors of ScNMT with potent IC50 values of 50 and 24 nM, respectively. In all three structures, the majority of the N-terminal region adopts a well defined structure with an {alpha}-helix and a loop located near the substrate-binding site and involved in the binding of MYA. The structural and kinetic data indicate that the N-terminal region plays an important role in the binding of both MYA and peptide substrate, but not in the subsequent catalytic reaction. Analyses of the interactions between the protein and the bound inhibitors also provide valuable information for further development of improved therapeutic agents.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Protein Expression and Purification—The ScNMT gene fragment corresponding to residues 3-455 was cloned into a customized expression plasmid (pBX3) that introduces a His tag (MRGSHHHHHHSMA) at the N terminus of the expressed protein. The recombinant plasmid was transformed into Escherichia coli strain BL21(DE3) pLysS (Novagen). Bacterial transformants were grown in LB medium supplemented with ampicillin (30 µg/ml) at 37 °C until A600 = 0.8, and then protein expression was induced with 0.2 mM isopropyl beta-D-thiogalactopyranoside for 6 h at 23 °C. The cells were harvested by centrifugation at 4500 x g and resuspended in buffer A (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM beta-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride). The cells were lysed on ice by sonication, and the insoluble cell debris was removed by centrifugation at 15,000 x g at 4 °C.

Protein purification was carried out by a combination of affinity chromatography and ion exchange chromatography at 4 °C. The lysis supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column (Qiagen Inc.) equilibrated with buffer A and then washed with buffer A supplemented with 5 mM imidazole. The target protein was eluted with buffer A supplemented with 100 mM imidazole. The eluted fractions were pooled together and dialyzed against buffer B (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM beta-mercaptoethanol, and 1 mM EDTA) for 3 h and then loaded onto a DEAE cation exchange column (Amersham Biosciences). The column was washed with buffer B, and the target protein existed in the flow-through fractions. SDS-PAGE and dynamic light scattering analyses indicated that the protein sample had high purity and homogeneity.

Expression plasmids of ScNMTs containing point mutations were generated from the vector containing the wild-type enzyme using the QuikChange site-directed mutagenesis kit (Stratagene). The sequences of these mutant genes were verified by DNA sequencing. Three ScNMT truncates were generated with deletion of residues 1-20 ({Delta}1-20), 1-30 ({Delta}1-30), and 1-40 ({Delta}1-40) of the N-terminal region, respectively. As a negative control, mutant ScNMT encoding the enzyme with deletion of C-terminal residues 452-455 ({Delta}452-455) was generated by mutating the codon specifying Val452 to a stop codon (TAG). Mutant ScNMT proteins were expressed and purified following the procedures used for the wild-type enzyme.

To perform the myristoylation enzymatic activity assay of ScNMT, an octapeptide (PARF1, GLFASKLF) corresponding to the N-terminal residues of the native substrate ARF1 of ScNMT was synthesized (Shanghai Science Peptide Biological Technology Co., Ltd.). The quality of the peptide was determined by analytical reverse-phase chromatography and mass spectral analysis and shown to have a purity of >95%.

Crystallization and Diffraction Data Collection—Crystallization was performed at 4 °C using the hanging-drop vapor diffusion method. MYA was dissolved in deionized water to a concentration of 15 mM, and the non-peptidic inhibitors were dissolved in Me2SO to a concentration of 25 mM.To prepare crystals of the binary complex of ScNMT with MYA, the ScNMT protein solution (~20 mg/ml) was first mixed with the MYA solution at a molar ratio of 1:1.5 and then incubated at 4 °C for 0.5 h before crystallization setup. Sheet-shaped crystals of the binary complex were grown in drops containing equal volumes (2 µl) of the protein mixture solution and the reservoir solution (20 mM HEPES (pH 7.1) and 2.6 M (NH4)2SO4) to a maximum size of 0.2 x 0.2 x 0.3 mm3 in 10 days. Diffraction data of the binary complex were collected to 2.9-Å resolution from a flash-cooled crystal at -176 °C at beamline 6A of the Photon Factory (Ibaraki, Japan) and processed with the HKL2000 suite (34). To prepare crystals of the ternary complex of ScNMT with MYA and non-peptidic inhibitors, the ScNMT protein solution was first mixed with the MYA and inhibitor solutions at a molar ratio of 1:1.5:5 and then incubated at 4 °C for 0.5 h before crystallization setup. Single crystals of the ternary complexes were grown under the same conditions as the binary complex to a maximum size of 0.15 x 0.15 x 0.2 mm3. Diffraction data of the ternary complexes were collected to ~3.0-Å resolution from flash-cooled crystals at -180 °C on an in-house Rigaku R-AXIS IV++ imaging plate detector equipped with a rotating anode generator. Diffraction data processing and scaling were performed using the CrystalClear suite (35). All of these complexes are of space group C2 and contain six ScNMT molecules in the asymmetric unit with a solvent content of ~50%. A summary of the diffraction data statistics is given in Table 1.


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  		TABLE 1
Summary of diffraction data and structure refinement statistics

 
Structure Determination and Refinement—The structure of the ScNMT·MYA binary complex was solved by molecular replacement with the program CNS (36) using the coordinates of ScNMT in complex with an MYA analog and a peptide substrate (Protein Data Bank code 1IID) (28) as the search model. The structures of the two ScNMT·MYA·inhibitor ternary complexes were determined by molecular replacement using the binary complex structure as the search model. Structure refinement was carried out with CNS following standard protocols (energy minimization, simulated annealing, and B factor refinement), and model building was facilitated using the program O (37). There are six ScNMT molecules in the asymmetric unit; thus, strict 6-fold non-crystallographic symmetry constraints were applied in the early stage of refinement, but released in the later stage of refinement. The final structure refinement was performed using the maximum likelihood algorithm implemented in the program REFMAC5 (38). A bulk solvent correction and a free R factor monitor (calculated with 5% of randomly chosen reflections) were applied throughout the refinement. The stereochemical quality of the structure models during the course of refinement and model building was evaluated with the program PROCHECK (39). The statistics of structure refinement are summarized in Table 1.

Myristoylation Enzymatic Activity Assay—The myristoylation activity of both wild-type and mutant ScNMTs was assayed by monitoring the production of CoA over time as described previously (40). The protein samples were diluted to 0.025 mg/ml (~0.5 µM) with HEPES buffer (0.1 M, pH 7.5), and the PARF1 octapeptide was diluted to 1 mM with HEPES buffer as a stock solution. The reaction solution consisted of 0.04 µM ScNMT, 0.16 mM MYA, and varying concentrations of PARF1 (0.16-0.80 mM with a concentration interval of 0.08 mM) in a total volume of 50 µl upon mixing with HEPES buffer. The reaction mixture was incubated at 37 °C for 1 h, and the reaction was stopped by the addition of 25 µl of 10 mM 5,5'-dithiobis(2-nitrobenzoic acid) in 0.1 M Tris-HCl (pH 8.0), 35 µl of 0.1 M Tris-HCl (pH 8.0), and distilled water to a total volume of 0.50 ml. The absorbance at 412 nm was recorded and blanked against an identical incubation sample without PARF1. The micromoles of CoA produced were calculated from a standard curve generated with known concentrations (0-200 mM) of beta-mercaptoethanol. The production of CoA was found to be linear over the 1-h time period of the assay, and product formation was a linear function of the amount of enzyme added. The kinetic parameters kcat and Km were obtained by fitting the data to the Michaelis-Menten equation with nonlinear regression. All experiments were repeated at least twice under the same conditions.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure of ScNMT—The crystal structures of ScNMT in binary complex with MYA and in ternary complexes with MYA and non-peptidic inhibitors Compounds I and II were solved by molecular replacement and refined to 2.9-, 3.1-, and 3.0-Å resolution, respectively (Fig. 1 and Table 1). The asymmetric unit contains six ScNMT molecules forming two pseudotrimers, which are almost identical with an average root mean square deviation of ~0.45 Å based on superimposition of all C-{alpha} atoms. As with other reported NMT structures, the enzyme has a compact saddle-shaped beta-sheet that spans the protein core surrounded by several {alpha}-helices. The NMT fold has an internal pseudo 2-fold symmetry with each half topologically equivalent to the monomer structure typical of a member of the GCN5-related N-acetyltransferase superfamily. In all three complex structures, there was very good electron density for the bound MYA and the majority of the N-terminal region of the enzyme in all six protomers of the asymmetric unit. In the structure with Compound I bound, there was good electron density for the inhibitor in each ScNMT molecule. However, in the complex with Compound II, there was good electron density for the inhibitor in only two ScNMT molecules, but poor density in the other four molecules. The model of the ScNMT·MYA complex comprises approximately residues 5-30 and 37-455 of each ScNMT molecule. The models of the ScNMT·MYA·Compound I/II complexes contain approximately residues 5-21 and 38-455 of each ScNMT molecule.

The overall structure of ScNMT in all three complexes (Fig. 2) is very similar to that described previously (27, 28). (Hereafter, the nomenclature of the secondary structure of ScNMT is that of Bhatnagar et al. (27).) However, a major difference involves the N-terminal region of the enzyme (residues 4-30). For the first time, it can be seen that this segment adopts a well defined conformation in all three of our complex structures, whereas the equivalent region is either disordered (27) or purposefully removed (28) in previous ScNMT structures. Interestingly, the N-terminal region (residues 1-60) is also invisible in the structures of C. albicans NMT both in the apo-form and in complexes with inhibitors (25, 26). The ordered N-terminal region in the ScNMT structures reported here forms an {alpha}-helix ({alpha}B') and loop (B'A') motif (Fig. 3). The {alpha}B' helix packs along a hydrophobic surface patch and appears to stabilize the overall structure. The B'A' loop is located on the surface of the protein near the MYA- and peptide-binding sites with interactions with the bound MYA and Ab loop. Another marked difference occurs in the Ab loop (residues 103-110), which adopts an open conformation in all three of our complex structures (Fig. 2B and Table 2), but assumes varying conformations in different structures reported previously (27, 28).


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  		TABLE 2
Comparison of the N-terminal region and the conformation of the Ab loop

 
Structure of the MYA-binding Site—Previous analyses of the crystal structures of ScNMT in binary complex with MYA and in ternary complexes with an MYA analog and a peptide substrate or peptidomimetic have identified structural elements involved in the binding of MYA, including helices {alpha}A' and {alpha}C; strands betae, betaf, and betan; and loops fg, eC, and Ab (27, 28). However, because of the absence of the N-terminal region of the enzyme in those complexes, full definition of the MYA-binding site appears to be incomplete. As described above, the majority of the N-terminal region is well resolved in our structures of ScNMT both in binary complex with MYA and in ternary complexes with MYA and Compounds I and II, which allows us to define the MYA-binding site more completely.


Figure 1
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  		FIGURE 1.
Overall structure of ScNMT. A, structure of the ScNMT·MYA binary complex. B, structure of the ScNMT·MYA·Compound I ternary complex. C, structure of the ScNMT·MYA·Compound II ternary complex. The N-terminal region of ScNMT (shown in gold) folds as an {alpha}-helix ({alpha}B') and a loop (B'A'). The MYA substrates are shown in ball-and-stick models in silver, and bound Compounds I and II are shown in magenta. D, SIGMAA-weighted Fo -Fc map (2{sigma} contour level) for bound Compounds I (left panel) and II (right panel) in their respective complex.

 
The MYA in our complexes assumes a "question mark" conformation similar to that observed in the ScNMT complexes reported previously. There are four bends at the pyrophosphate group, the C-6 - C-7 positions of pantetheine, the C-1 position of myristate, and the C-5-C-6 positions of myristate, respectively. In our ScNMT·MYA complex, MYA maintains the interactions with residues of the structural elements described previously (27, 28). In addition, the N-terminal region also forms part of the MYA-binding site, and several residues of the B'A' loop are directly or indirectly involved in the binding of MYA (Fig. 3). Specifically, the side chain N-{epsilon} atom of Gln29 forms a hydrogen bond with the 2'-hydroxyl group of the ADP-ribose moiety (2.8 Å); the side chain N-{delta} atom of His38 forms a hydrogen bond with one 3'-phosphate oxygen of ADP (2.6 Å); and the side chain N-{epsilon} atom of His38 forms a hydrogen bond with the side chain O-{epsilon} atom of Glu11 (2.6 Å). The aromatic side chain of Phe27 makes {pi}-{pi} stacking interaction with the adenine moiety of ADP. These interactions further stabilize the binding of MYA. Moreover, the B'A' loop has several hydrophilic interactions with the Ab loop, which was also suggested to play a critical role in the binding of MYA (27, 28). The main chain carbonyl of Lys26 forms a hydrogen bond with the side chain O-{epsilon} atom of Glu105, and the side chain O-{gamma} atom of Ser25 forms hydrogen bonds with the main chain amide of Glu105 and main chain carbonyl of Tyr103. These interactions appear to stabilize the conformation of the Ab loop and its interactions with MYA. It is note-worthy that omission of MYA in the crystallization solution still resulted in the structure of ScNMT with bound MYA and a well resolved N-terminal region, indicating that MYA has a very tight binding affinity with the enzyme and can be co-purified with the enzyme from the expression system and that the presence of the N-terminal region might stabilize the binding of MYA.

Structure of the Non-peptidic Inhibitor-binding Site—The non-peptidic inhibitors Compounds I and II are bound at the peptide-binding site and have primarily hydrophobic interactions with the surrounding residues (Fig. 4). Structural comparisons indicate that the overall structures of the inhibitor-bound complexes are similar to that of the ScNMT·MYA complex and that binding of the inhibitors does not induce substantial conformational change in the peptide-binding site, except minor adjustment of the side chains of a few residues. These results suggest that this site has a rigid structure and that the inhibitor has to have proper structural and chemical properties to bind most effectively. Analysis of the chemical properties of the inhibitors indicated that Compound II is more hydrophobic than Compound I, consistent with the former having a relatively higher ClogP value (5.42 for Compound II versus 3.62 for Compound I). Structure comparison showed that Compound II binds slightly deeper toward the catalytic active site and has more hydrophobic contact with surrounding residues than Compound I (Fig. 4). Binding of Compound II with the protein buries 70.0% (393.8 Å2) of the surface area of the inhibitor, whereas binding of Compound I with the protein buries 53.8% (295.5 Å2) of the surface area of the inhibitor.

In the ScNMT structure with bound Compound I, the inhibitor has mainly hydrophobic interactions with a number of aromatic residues, including Tyr103, Phe111, Phe113, Tyr219, Phe234, Phe334, and Tyr349 (Fig. 4A). The benzene ring of the inhibitor stacks perpendicularly with the aromatic side chain of Phe234, whereas the thiazolidine moiety interacts with Phe111, Phe113, Phe334, and Tyr349. The nitrophenol moiety is located at the center of the catalytic active site and points toward the carboxylate group of the C-terminal Leu455 residue (~7.5 Å) and the thioester carbonyl group of MYA (~8.0 Å). It is also well positioned to make both hydrophobic and hydrophilic interactions (possibly via water molecules) with several residues nearby, including Tyr103, Phe113, Gly207, Tyr219, Leu455, and the pantetheine group of CoA.

His221 and Asp417 have been implicated to be crucial in binding of the peptide substrate. Steady-state kinetic studies have shown that His221 plays an important role in enzymatic activity and that mutation H221A incurs a marked increase in Km (41). Biochemical data have also shown that this mutant is unable to support yeast survival (42). In the structure of ScNMT in complex with a peptide or peptidic inhibitor, the imidazole ring of His221 forms a hydrogen bond with the hydroxyl group of Ser at position +5 of the peptide or related inhibitor (27, 28). In the structure of C. albicans NMT in complex with a non-peptidic inhibitor, the side chain of His221 also makes a hydrogen bond with the benzofuran ring of the inhibitor (26). In the Compound I-bound ternary complex, both His221 and Asp417 point their side chains toward, but have no direct interaction with, the inhibitor. However, there are several spherical residual electron density peaks that are located between these residues and the 2'-thionyl group of the thiazolidine moiety of the inhibitor. These peaks could be water molecules, which might mediate the interaction between these two residues and the inhibitor, stabilizing inhibitor binding. Clearly, this is a feature of the inhibitor that might be altered to further enhance the hydrophilic interaction with the enzyme and to improve binding affinity. In addition, the 4'-carbonyl group of the thiazolidine moiety has no contact with the surrounding residues in the hydrophobic environment. Thus, substitution of this polar group with a hydrophobic group might improve its interactions and thus binding. Moreover, additional groups on the nitrophenol moiety might also prove beneficial.


Figure 2
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  		FIGURE 2.
Comparison of different NMTs. A, structure-based sequence alignment of ScNMT with representative NMTs from other species. CaNMT, C. albicans NMT; PiNMT, Phytophthora infestans NMT; HsNMT1 and HsNMT2, H. sapiens NMT types 1 and 2, respectively. Strictly conserved residues are highlighted in shaded red boxes, and conserved residues in open red boxes. The secondary structure of ScNMT is shown at the top of the alignment. The sequence alignment was generated using ESPript (48). B, structural comparison of ScNMT in different complexes. The structure of ScNMT in binary complex with MYA described here is shown in yellow; the structure of ScNMT in ternary complex with an MYA analog and a peptide substrate (Protein Data Bank code 1IID) is shown in cyan; and the structure of ScNMT in ternary complex with an MYA analog and SC-58272 (Protein Data Bank code 2NMT) is shown in green. A marked conformational difference among these three ScNMT complex structures occurs in the Ab loop region. C, superposition of the three ScNMT complexes showing the conformational difference of the Ab loop. The Ab loop adopts a closed conformation in the structure of the ScNMT complex with an MYA analog and SC-58272 (green). Asp106 and Asp108 of the Ab loop make close contacts with the Lys residue at position +6 of the peptide substrate. In the structure of the ScNMT complex with an MYA analog and a peptide substrate, the Ab loop assumes a semi-open conformation (cyan) and has no direct contact with the peptide substrate-binding site. In the ScNMT complex with MYA described here, the Ab loop adopts an open conformation (yellow) and has no contact with the peptide substrate-binding site, too. Instead, the side chains of Asp22, Asp23, and Thr24 of the N-terminal region occupy the corresponding positions of Asp106 and Asp108 of the Ab loop in the closed conformation and appear to be involved in the recognition and binding of the Lys residue at position +6 of the peptide substrate. D, electrostatic surfaces of ScNMT in different complexes showing the Ab loop in closed conformation (left panel), semi-open conformation (middle panel), and open conformation (right panel). The peptide-binding site is covered by residues of the Ab loop in the closed conformation and is exposed to the solvent in the semi-open conformation, but is partially covered by the N-terminal region in the open conformation.

 
In the Compound II-bound ternary complex, the inhibitor makes mainly hydrophobic contacts with aliphatic residues Leu332, Ile347, and Val395 and aromatic residues Tyr103, Phe111, Phe113, Tyr115, Phe119, Tyr219, Phe234, Tyr330, Phe334, and Tyr349 (Fig. 4B). The methyltetrahydrocarbazole moiety of the inhibitor has hydrophobic interactions with Tyr219, Phe234, and Val395. In addition, both His221 and Asp417, which are involved in the recognition and binding of the peptide substrate, make hydrophobic rather than hydrophilic interactions with the cyclohexane ring. The amine group of the inhibitor is in a position to form a hydrogen bond with the hydroxyl group of Tyr349. In addition, the adjacent hydroxyl group also makes a hydrophilic interaction with the hydroxyl group of Tyr349. The terminal cyclohexane ring of Compound II is surrounded by a number of aromatic residues, including Tyr103, Phe113, Tyr115, Phe119, Tyr330, and Tyr349. It is located in proximity to the carboxylate group of Leu455 (~3.5 Å). Because there is some room between the cyclohexane ring of the inhibitor and Leu455, modification of the ring to augment its interaction with the C-terminal Leu455 residue might further stabilize inhibitor binding.

Functional Roles of the N-terminal Region of ScNMT—In all previously reported structures of NMTs from S. cerevisiae and C. albicans, the N-terminal region of the enzyme is either disordered or removed during structural studies (25-28). In the ScNMT structures reported here, the majority of the N-terminal region is defined and folds as an {alpha}-helix ({alpha}B') and a loop (B'A'). Analysis of our ScNMT complexes and comparison with other S. cerevisiae and C. albicans NMT structures allow us to discuss, for the first time, the potential functional roles of the N-terminal region of NMT.


Figure 3
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  		FIGURE 3.
Structure of the N-terminal region of ScNMT. A, molecular surface of ScNMT near the MYA-binding site in binary complex with MYA reported previously (28) (left panel) and described here (right panel). The N-terminal region defined in our structure (shown in gold) interacts with residues of the MYA-binding site and appears to stabilize the overall structure of the enzyme. B, stereo view showing the interactions of the N-terminal region with MYA and residues of the Ab loop. It seems possible that the N-terminal region might participate in the recognition and binding of both MYA and the peptide substrate.

 
Structural comparison showed that the Ab loop (residues 103-110) adopts different conformations (open, semi-open, and closed) in the previously reported NMT structures (Fig. 2B and Table 2). In the structure of ScNMT in complex with an MYA analog and the peptidic inhibitor SC-58272, the Ab loop assumes a closed conformation, and residues of the Ab loop (Val104-Gly110) cover the peptide-binding site and interact with the peptide substrate (27). In particular, the charged {epsilon}-amino group of the crucial Lys residue at position +6 is tightly surrounded by the side chains of acidic residues Asp106, Asp108, and Asp417 (Fig. 2C). These results led to the suggestion that the Ab loop plays an important role in the recognition and binding of the peptide substrate (27). However, in the structure of ScNMT complexed with only MYA, the Ab loop assumes an open conformation in one monomer and a semi-open conformation in the other (28). In the open conformation, the Ab loop is pointed toward the solvent, and the tip of the loop is ~8 Å away from the peptide-binding site. The semiopen conformation is between the closed and open conformations, with the tip of the Ab loop being ~5 Å away from the peptide-binding site. Also, in the structure of ScNMT in complex with an MYA analog and a peptide substrate, the Ab loop adopts a semi-open conformation rather than the closed conformation, and residues of the Ab loop have no interaction with the side chain of the crucial Lys residue of the peptide (28). In the structures of C. albicans NMT, the Ab loop assumes a closed conformation in its complex with MYA and SC-58272, but is disordered in the apo-form and in its complex with a non-peptidic inhibitor (25, 26). Interestingly, in all three of our ScNMT complex structures, the Ab loop assumes the open conformation with well defined electron density and has no interaction with the bound non-peptidic inhibitors. These results indicate that the Ab loop has a high flexibility and is unlikely to be directly involved in the recognition and binding of the peptide substrate. Moreover, sequence comparison also indicated that most of the residues forming the Ab loop are strictly conserved in all known NMTs from different species and that these conserved residues, such as Asp106 and Asp108, are less likely to participate in the recognition of the variable sequences of species-specific peptide substrates (Fig. 2A).


Figure 4
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  		FIGURE 4.
Structure of the non-peptidic inhibitor-binding site. A, interactions between Compound I and the surrounding residues. B, interactions between Compound II and the surrounding residues. C, superposition of the inhibitor-binding site in the Compound I-bound (green) and Compound II-bound (cyan) ternary complexes. Structural comparison indicated that the inhibitor-binding site has a relatively rigid structure and that binding of the inhibitor induces very minor conformational changes in the residues involved in interactions. Compound II is more hydrophobic than Compound I and is bound slightly deeper toward the catalytic active site.

 
On the other hand, detailed analysis of our ScNMT structures showed that the B'A' loop of the N-terminal region takes the position of the Ab loop in the closed conformation and has interactions with the bound inhibitors (Fig. 2). In particular, Asp22, Asp23, and Thr24 of the B'A' loop occupy the corresponding positions of Asp106 and Asp108 of the closed Ab loop and are well positioned to interact with the side chain of Lys at position +6 of the peptide substrate, suggesting that these residues might be involved in the recognition and binding of the peptide substrate. Sequence comparison also showed that the residues forming the {alpha}B' helix are moderately conserved in most NMTs from different species, but that the residues forming the B'A' loop are not conserved (Fig. 2A), suggesting that the B'A' loop might be the determinant of species-specific function(s).

To investigate further the functional roles of these various residues and the N-terminal region in the myristoylation reaction, we carried out mutagenesis studies and truncation analysis and determined the kinetic parameters of the altered ScNMTs (Table 3). Both single and double mutations of Asp106 and Asp108 to Ala or Lys in the Ab loop had no significant effect on the Km value. On the other hand, whereas single mutations of Asp22, Asp23, and Thr24 to Ala or Lys had minor effects on the Km value, double mutations D22A/D23A and D22K/D23K caused a substantial increase in Km (~7- and 11-fold, respectively). Consistently, truncation of the first 30 or 40 residues of the N-terminal region, which removed Asp22 and Asp23, caused an ~12-fold increase in Km, whereas truncation of the first 20 residues had a much smaller effect (2-fold). However, none of these mutations had any significant effect on the kcat value. These data further substantiate our structural results that Asp106 and Asp108 of the Ab loop are not involved in the binding of the peptide substrate, whereas Asp22 and Asp23 of the N-terminal B'A' loop are involved in the binding of the peptide substrate, but do not participate in catalysis, consistent with previous biochemical data showing that removal of the N-terminal 34 residues of ScNMT has no significant effect on enzymatic activity (43).


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  		TABLE 3
Kinetic data for myristoylation of PARF1 by wild-type and mutant ScNMTs

 
Together, our structural and kinetics data indicate that Asp106 and Asp108 of the Ab loop are not directly involved in the recognition and binding of the peptide substrate. Instead, Asp22 and Asp23 of the N-terminal B'A' loop assume this role. These two residues appear to provide the negatively charged electrostatic environment to recognize and bind the positively charged side chain of the lysine residue at position +6 of the peptide substrate. Thus, the closed conformation of the Ab loop seen in the ScNMT·MYA analog·inhibitor complex (27) could be an artifact resulting from the absence of the N-terminal region.

In addition to the involvement in the recognition and binding of the peptide substrate, the N-terminal region of ScNMT appears to have other functional role(s). As discussed above, the N-terminal region forms part of the MYA-binding site. In particular, several residues of the B'A' loop have both hydrophilic and hydrophobic interactions with MYA and help to stabilize the Ab loop of the enzyme, which is also involved in MYA binding. Thus, it is evident that the N-terminal region plays an important role in the recognition and binding of MYA. Structural comparison also showed that, in the absence of the N-terminal region, Phe49, Ile208, Val209, Pro213, and Phe419 form a hydrophobic patch near the MYA-binding site that is directly exposed to the solvent, conceivably making the enzyme less stable. With the N terminus in place, the {alpha}B' helix covers this hydrophobic patch and forms both hydrophobic and hydrophilic contacts with the rest of the protein. Therefore, it is very likely that the N-terminal region helps to stabilize the overall structure of the enzyme.

Moreover, protein N-myristoylation appears to be a tightly regulated reaction involving coordinated participation of several different enzymes/proteins (e.g. N-methionyl aminopeptidase, fatty-acid synthetase, long chain acyl-CoA synthetase, acyl-CoA-binding proteins, etc.), access of NMT to pools of MYA, and timely N-myristoylation of nascent polypeptide substrates to avoid potential interfering reactions (e.g.N-acetylation and polypeptide folding) (for reviews, see Refs. 44-47). The ability of NMT to function within such a complicated process implies the existence of mechanisms designed to ensure targeting of the enzyme to the appropriate protein synthesis machinery, possibly involving interactions with other cooperating components that facilitate the recognition and efficient N-myristoylation of the rapidly growing polypeptide substrates. However, so far, no information is available regarding the mechanism(s) regulating either the specific association of NMT with the cytoplasmic protein synthesis machinery or its direct participation during protein synthesis. Both are presumably required for ScNMT to accomplish the co-translational N-myristoylation of proteins in yeast cells. Some experiments have shown that the N-terminal region of human NMT plays an important role in targeting the enzyme to the site of protein synthesis on ribosomes, thereby facilitating the participation of the enzyme in the co-translational N-myristoylation of proteins in mammalian cells (29). Because the N-terminal region of ScNMT is located on the molecular surface near both the MYA- and peptide-binding sites, it is possible that the N-terminal region and other structural elements might provide a potential binding site for other associated proteins in the subcellular localization of the enzyme and thus coordinated control of its catalytic activity.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2P6E, 2P6F, and 2P6G) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The work performed in the Chinese Academy of Sciences was supported by Grants 2004CB720102, 2006CB806501, and 2006AA02Z112 from the Ministry of Science and Technology of China and by Grant 30570379 from the National Natural Science Foundation of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: DuPont Stine Haskell Research Center, 1090 Elkton Rd., Newark, DE 19714. Tel.: 302-366-6558; Fax: 302-366-5738; E-mail: Steven.Gutteridge{at}usa.dupont.com.

1 To whom correspondence may be addressed. Tel.: 86-21-5492-1619; Fax: 86-21-5492-1116; E-mail: jpding{at}sibs.ac.cn.

3 The abbreviations used are: NMTs, myristoyl-CoA:protein N-myristoyltransferases; MYA, myristoyl-CoA; Sc, S. cerevisiae. Back


   ACKNOWLEDGMENTS
 
We are grateful to the staff members of the Photon Factory for technical support in data collection, Vern Wittenbach and Wayne Hanna for technical assistance, and other members of our groups for helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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