Structural analysis of inhibition of E. coli methionine aminopeptidase: implication of loop adaptability in selective inhibition of bacterial enzymes
© Ma et al; licensee BioMed Central Ltd. 2007
Received: 26 June 2007
Accepted: 19 December 2007
Published: 19 December 2007
Methionine aminopeptidase is a potential target of future antibacterial and anticancer drugs. Structural analysis of complexes of the enzyme with its inhibitors provides valuable information for structure-based drug design efforts.
Five new X-ray structures of such enzyme-inhibitor complexes were obtained. Analysis of these and other three similar structures reveals the adaptability of a surface-exposed loop bearing Y62, H63, G64 and Y65 (the YHGY loop) that is an integral part of the substrate and inhibitor binding pocket. This adaptability is important for accommodating inhibitors with variations in size. When compared with the human isozymes, this loop either becomes buried in the human type I enzyme due to an N-terminal extension that covers its position or is replaced by a unique insert in the human type II enzyme.
The adaptability of the YHGY loop in E. coli methionine aminopeptidase, and likely in other bacterial methionine aminopeptidases, enables the enzyme active pocket to accommodate inhibitors of differing size. The differences in this adaptable loop between the bacterial and human methionine aminopeptidases is a structural feature that can be exploited to design inhibitors of bacterial methionine aminopeptidases as therapeutic agents with minimal inhibition of the corresponding human enzymes.
Methionine aminopeptidase (MetAP) removes the N-terminal methionine residue from nascent proteins in all types of cells . Prokaryotic cells express only one MetAP, and its essentiality was demonstrated by the lethality of its deletion from Escherichia coli  and Salmonella typhimurium . MetAP is therefore a potential target for developing novel broad spectrum antibacterial drugs . Eukaryotic cells have two types of MetAP (type I and type II), and deletion of both MetAP genes in Saccharomyces cerevisiae was shown to be lethal [5, 6]. Fumagillin and its analogues TNP-470 and ovalicin are potent antiangiogenic compounds and are also selective inhibitors of human type II MetAP [7–9]. The antiproliferative bengamides inhibit both types of human MetAP . Therefore, human MetAPs may also serve as targets for development of new anticancer therapeutics.
Early MetAP inhibitors were derived from peptide substrates or the cleavage product methionine, such as the peptic inhibitor (3R)-amino-(2S)-hydroxyheptanoyl-L-Ala-L-Leu-L-Val-L-Phe-OMe (Ki 5 μM)  and norleucine phosphonate (NleP) . Both are considered as transition state inhibitors. Although these compounds are not desired as therapeutic agents, structural studies of their complexes with MetAP have provided valuable insight of the catalysis and inhibition of MetAP [12–14]. Fumagillin, a natural product, and its analogues are a unique class of MetAP inhibitors that covalently modify a conserved histidine residue at the active site (H79 of E. coli MetAP, and the equivalent H231 of human type II MetAP) [9, 15, 16]. Several classes of non-peptidic and reversible MetAP inhibitors have been identified recently, such as furancarboxylic acids [17, 18], thiabendazole and other thiazole-containing compounds [17, 19–21], triazole-based derivatives [22–24], and sulfonamides [25, 26]. However, structural analysis of these nonpeptidic inhibitors in complex with MetAP showed that inhibition by many of the thiazole and triazole-containing compounds and sulfonamides is metal-mediated, and they bind to the active site of enzyme through a divalent metal ion with one of the conserved active site histidines (most with H97, and some with H181; both are E. coli MetAP numbering) [19, 21, 25]. It has been pointed out that formation of such complexes may be an artefact during crystallization or in in vitro assays using high metal concentrations [14, 19, 27], and whether there are enough free metal ions available inside cells to form such inhibitor-enzyme complexes is a question.
MetAP was initially characterized as a Co(II) enzyme because of reproducible activation of the apoenzyme by Co(II) [5, 28]. Many X-ray structures of MetAPs with or without a ligand bound  show a dinuclear metal site inside the active site pocket that has five conserved residues D97, D108, H171, E204 and E235 (E. coli MetAP numbering) as metal ligands and filled with two Co(II) ions. The metal ion used to form the inhibitor-enzyme complexes mentioned above is neither of the metal ions, but an additional one close to the dinuclear site. In addition to Co(II), other divalent metals such as Mn(II), Ni(II), Zn(II), and Fe(II) have been shown to activate the enzyme in vitro as well [30, 31]. It is not known which of the metal ions is actually used by MetAP under physiological conditions, but speculation favors Fe(II), Zn(II) or Mn(II) for this role [23, 31, 32].
Results and Discussion
Overall structure of E. coli MetAP in complex with the inhibitors
X-ray data collection and refinement statistics
X-ray Data Collection
Resolution range (Å)
Completeness (%) a
Rmerge (%) a
RMSD bonds (Å)
RMSD angles (deg)
No. of solvent molecules
<B> enzyme (Å 2)
<B> inhibitor (Å 2)
<B> water (Å 2)
Binding of the inhibitors to E. coli MetAP
Adaptability of the YHGY loop in the inhibitor binding pocket
rmsd values generated by pair-wise comparisons of the eight complex structuresa
Statistical analysis of the loop adaptability
Although the eight structures show good overall alignment as indicated by their small rmsd values during pair-wise comparisons (Table 2), the YHGY loop assumes different positions when different inhibitors are present (Fig. 4). It is revealing that when the residues 61–64 (the YHGY loop) are excluded from the rmsd calculation, the values dropped significantly for some of the pairs (Table 2), indicating a major contribution of the YHGY loop to the rmsd values for these pairs. Notably, the rmsd value between 1XNZ (complex with 1) and 2Q96 (complex with 8) showed the biggest drop from 0.356 Å to 0.304 Å with a reduction of 0.051 Å (14%) just by removing four out of 253 residues for the rmsd calculation.
Comparison with the structures of human type I and type II MetAPs
By overlaying the structures of E. coli MetAP and human type I MetAP, we see that the N-terminal extension of the human enzyme wraps around the enzyme surface and covers the YHGY loop (Fig. 7A). The surface-exposed loop in E. coli MetAP now becomes mostly buried in human type I enzyme. This could greatly reduce the plasticity of the loop and make the inhibitor binding pocket much less tolerant to structural variations in inhibitor molecules. This change is consistent with our previous observation on inhibition of the truncated and full length human type I MetAPs , and the partially buried nature of the loop could make it less adaptable for the inhibitors of differing size.
In addition, an overlaying of the E. coli MetAP and human type II MetAP structures reveals that the YHGY loop is not present at all in human type II MetAP, and instead, its position is now occupied by an insert, unique to type II MetAP (Fig. 4B). The insert of approximately 65 residues forms a distinct globular domain and is an integral part of the active site pocket. The adaptability of its binding pocket may be analyzed when more complex structures are available. However, differences in adaptability are likely found because the YHGY loop is part of the pocket in E. coli MetAP and it is substituted by the globular insert in human type II MetAP. It is interesting to note that the N-terminal extension of human type II enzyme is located away from the active site.
Implications of the adaptability of the YHGY loop in developing MetAP inhibitors as antibiotics
Structure-based drug design takes advantage of the structural information of a medicinally important protein, especially at the active site, to guide the design and development of protein ligands to achieve desired potency and selectivity. Bacterial MetAP enzymes are the simplest in the MetAP family and contain only a catalytic domain. Most bacterial MetAPs, with exception of achaeal enzymes, belong to the type I MetAP family and are homologs. E. coli MetAP, as a typical bacterial MetAP, has high sequence homology with human type I MetAP within the catalytic domain (121 out of 264 residues are identical). It is certainly desirable to identify the differences between bacterial MetAPs and human counterparts so that MetAP inhibitors as potential antibiotics will selectively inhibit only bacterial MetAPs. The N-terminal extension and the insert in human MetAPs are the extra structural elements that can be potentially exploited to design selective inhibitors for bacterial MetAPs.
As more X-ray structures of E. coli MetAP in complex with inhibitors have become available, we now can compare the structures and characterize the binding of different inhibitors. Careful structural analysis of these structures reveals the adaptability of the YHGY loop (Y62, H63, G64 and Y65) that accommodates inhibitors of differing size. The adaptability of this loop in bacterial enzymes could be an important structural feature to exploit because the loop is partially occluded by the N-terminal extension in human type I MetAP [38–42] and is replaced by an unique insert in human type II MetAP . Consequently, the ability to adapt multiple conformations within this loop of E. coli MetAP, and likely of other bacterial MetAPs, may not exist in human MetAPs and could be utilized to steer MetAP inhibitors towards selective inhibition of the bacterial enzymes.
This observation emphasizes the importance in considering the dynamics of ligand binding to enzymes in modeling inhibitors into a binding site on a protein, especially during the virtual screening of MetAP inhibitors. A rigid active site would appear to be relatively intolerant of anything but a nearly perfect fit or a slightly undersized ligand, but a flexible site would be more forgiving and tolerant of a wider range of structures. Molecules that might appear to fit poorly based on a rigid structure model may in reality fit quite well because of loop movement. Recognizing and utilizing this flexibility could be beneficial, for example, for optimizing the potency and selectivity of an inhibitor or fine-tuning its biopharmaceutical properties.
Structural analysis of the complexes of E. coli MetAP with a series of related inhibitors reveals the ability of the surface-exposed loop containing the sequence YHGY to adapt multiple conformations to better complement the structural features of bound ligands. This adaptable loop likely exists in all bacterial MetAPs based on sequence similarity and the surface-exposed nature of the loop. However, this loop is partially buried by an N-terminal extension in the human type I MetAP and substituted by a globular insert in the human type II MetAP. The difference in ability of the substrate/inhibitor binding pocket to adapt to a wide range of ligand sizes may distinguish bacterial MetAPs from human MetAPs and could be exploited to design selective inhibitors of bacterial MetAPs.
Preparation of the protein and compounds
The recombinant E. coli MetAP was purified as an apoenzyme . Compounds 1–3, 6 and 7 were purchased from ChemBridge (San Diego, CA) and characterized by 1H and 13C NMR and high resolution mass spectrometry. Compounds 4, 5 and 8 were synthesized in our laboratory. Their inhibitory activities on the Co(II)-, Mn(II)-, Ni(II)- and Fe(II)-forms of E. coli MetAP have been described previously [17, 18].
Initial crystallization conditions were determined using Crystal Screen and Index HT kits in 96-well sitting-drop plates (Hampton Research) at room temperature. Final crystals of the enzyme-inhibitor complexes were obtained independently by the hanging-drop vapour-diffusion method at 18–20°C. Inhibitors (200 mM in DMSO) were added to concentrated apoenzyme (12 mg/ml, 0.4 mM) in 10 mM MOPS pH 7.0. Hanging drops contained 3 μl protein solution mixed with 3 μl reservoir solution. The reservoir solution consisted of 10–15% PEG 20,000, 0.1 M MES (pH 6.5) and 0.2 mM MnCl2. The concentration ratio of inhibitor:apoenzyme was 5:1 for 4 and 5 and 10:1 for 6–8, and that of metal:apoenzyme was 5:1.
Data collection and structural refinement
Data were collected on an R-Axis IV imaging plate detector with a Rigaku rotating anode generator operated at 50 kV and 100 mA. Images were recorded over 180° in 0.5° increments at 100 K. Raw reflection data were indexed and integrated using MOSFLM  and merged and scaled using SCALA in CCP4  with CCP4i interface . Analysis of the estimated solvent content of each crystal  indicated only one molecule of the enzyme per asymmetric unit in all cases. The coordinates of our previously solved structure of E. coli MetAP (PDB code 1XNZ) with ligand, metal ions and water molecules removed were used as the search model for molecular replacement using MOLREP . Crystallographic refinement was performed with CNS . The refinement was monitored using 10% of the reflections set aside for free R factor analysis throughout the whole refinement process. Initial refinement started with simulated annealing with a starting temperature at 4000 K and 25 K drop in temperature per cycle. The models were refined with iterative cycles of individual B factor refinement, positional refinement, and manual model building using WinCoot . The Mn(II) atoms were not included in the initial refinement procedure to reduce the model bias in phases and were then added to the model to the center of the peak in the Mn(II)-omitted Fobs-Fcalc electron density map. The ligand and water molecules were added when the electron densities shown in 2Fobs-Fcalc and Fobs-Fcalc maps for their placement were unequivocal. The final 2Fobs-Fcalc maps showed clear electron density for most of the atoms except for a few side chains at the molecular surface. The final models for all of the structures were analyzed using the program PROCHECK , and all have 99.6% of residues were in the allowed region of their respective Ramachandran plots. The atomic coordinates and structure factors for the structures have been deposited in the Protein Data Bank. Statistic parameters in data collection and structural refinement are shown in Table 1.
Structures were aligned with PYMOL  using the "align" command, and rmsd values were calculated with "rms" command after pair-wise alignment. The program ESCET [35, 36] was used to make an objective analysis of the conformational variability of the eight structures 1XNZ, 2EVM, 2EVC, 2Q92, 2Q93, 2Q94, 2Q95 and 2Q96. All drawings for protein structures in the figures were generated using PYMOL.
We thank Dr. Min Huang and Dr. Robert P. Hanzlik for helpful discussions, Dr. Wei-Jun Huang for his assistant in X-ray data collection, and Dr. Thomas Schneider for his program ESCET used in calculating error-scaled difference distance matrices. This research was supported by NIH Grants AI065898, RR015563 and RR016475 (to QZY). High Throughput Screening Laboratory and Protein Structure Laboratory were supported by NIH Grants RR015563 and RR017708 from COBRE program of National Center for Research Resources, University of Kansas, and Kansas Technology Enterprise Corporation.
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