Molecular Modeling and Active Site Binding Mode Characterization of Aspartate β-Semialdehyde Dehydrogenase Family.

DOI: 10.1002/minf.201200128

Molecular Modeling and Active Site Binding Mode Characterization of Aspartate b-Semialdehyde Dehydrogenase Family Rajender Kumar[a] and Prabha Garg*[a, b]

Abstract: The enzyme aspartate b-semialdehyde dehydrogenase (ASADH) plays a vital role in biosynthesis of essential amino acids and several important metabolites in microbes and some higher plants. So this key enzyme can be targeted selectively in these microbes to exhibit anti-bacterial and fungicidal effects. In this work, molecular modeling and comparative active site binding mode studies were performed for understanding the mode of action, in silico insight into the 3D structure, enzyme-substrate interactions with natural substrate in this homologous enzyme family.

During comparative sequence analysis, high diversity was found in the sequences of different ASADHs and exhibited the same key binding interactions with the substrate. Both, the functional carboxylic and the phosphate group of the substrate are engaged in a bidentate interaction with the guanidinium N atom of two key arginyl active site residues of ASADHs. These structural and active site binding mode characterization studies can further be used for designing the more potent and selective substrate analogues inhibitors against ASADH family.

Keywords: Aspartate b-semialdehyde dehydrogenase · Comparative sequence analysis · Clustering · Molecular modeling · Docking

1 Introduction Aspartate b-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme for the biosynthesis of essential amino acids in prokaryotes, fungi, and some higher plants. ASADH is not present in the mammals.[1] In the aspartate biosynthetic pathway, ASADH catalyzes the reductive dephosphorylation of b-aspartyl phosphate (bAP) to l-aspartate b-semialdehyde (ASA).[2] From this point, one branch leads to the production of lysine through the metabolite diaminopimelate, while the another branch leads to the product ASA which is further reduced to homoserine and then converted to the amino acids threonine, isoleucine and methionine. Several important metabolites are synthesized in the aspartate pathway that plays fundamental roles in important developmental processes, such as cell wall biosynthesis, virulence factor production, etc. Dipicolinate is a major component of bacterial spores,[3] and diaminopimelate (DAP) is required for cross-linking of the peptidoglycan polymers in bacterial cell wall synthesis.[4] An additional product of this pathway, S-adenosylmethionine serves as a precursor for quorum sensing signaling molecules with vital roles in triggering virulence factors in infectious microbes.[5,6] The knockout as well as perturbations to the asd gene have been reported lethal in Legionella pneumonphila,[7] Salmonella typhimurium[8] and Streptococcus mutans[9] due to the auxotroph for DAP, an intermediate metabolite in the aspartate pathway. Inhibition of this biosynthesis pathway is a promising strategy for the development of novel antibiotic, fungicidal, or herbicidal agents.[1] Selective inhibition of this key enzyme can produce lead Mol. Inf. 2013, 32, 377 – 383

compounds that could play a role in combating the growing threat from multidrug-resistant infectious organisms. The 3D crystal structure of this essential enzyme has been determined from a wide variety of microbes, including Gram- negative[10–12] and Gram-positive bacteria[13] , fungal[14] and archael species.[15] The catalytic mechanism of ASADH is supported by kinetic studies,[16,17] mutagenesis studies[18–20] and structural characterization of numerous key catalytic intermediates.[10,13] The molecular level characterization and understanding of the structural and mechanism of ASADH is being used to guide the development of novel selective enzyme inhibitors. Various substrate analogous inhibitors were already reported for ASADH target.[21–25] The first synthetic inhibitor for ASADH was reported as difluoromethylene analogues of aspartyl phosphate which has shown good binding inhibition.[22] Molecular modeling and docking studies have identified new chemical entities that were tested against repre[a] R. Kumar, P. Garg Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER) S. A.S. Nagar, Punjab 160062, India tel.: + 91-172-2292016; fax: + 91-172-2214692 *e-mail: [email protected] [b] P. Garg Computer Centre, National Institute of Pharmaceutical Education and Research (NIPER) S.A.S. Nagar, Punjab 160062, India

2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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sentative ASADHs. These docking models were also used for virtual screening of a small library of commercially available structures to identify compounds with a high potential for selective ASADH inhibition.[26] The objective of this study is comparative sequence, structural and active site binding mode characterization of the ASADH enzyme family using computational techniques. This study can further be used to rational drug designing for the potential drug target ASADH in various pathogens species.

2 Computational Methods In this study, twelve infectious organisms were selected: Vibrio cholerae (vcASADH 1 and vcASADH 2), Bacillus subtilis (bsASADH), Neisseria meningitidis (nmASADH), Campylobacter jejuni (cjASADH), Escherichia coli (ecASADH), Haemophilus influenzae (hiASADH), Helicobacter pylori (hpASADH), Mycobacterium tuberculosis (mtASADH), Pseudomonas aeruginosa (paASADH), Salmonella typhi (stASADH), Candida albicans (caASADH) and Streptococcus pneumonia (spASADH). The primary protein sequences of ASADHs were retrieved from Uniport Knowledgebase release 15.12. Reported 3D structures of ASADHs were taken from the Protein Data Bank (PDB). The detail information is given in table 1. The homology models of ASADH proteins were built using the Modeller9v[27] for which 3D crystal structure were not reported in PDB. The NCBI BlastP was performed against the PDB for identification of highly similar sequence templates for query sequences. After aligning with the help of Align2D script, the query and template sequences were used as input in the program Modeller and homology models were generated for ASADH proteins of six species. Modeled structures often produces unfavourable bond lengths, bond angles, torsion angles and contacts. Therefore, it was essential to minimize the energy to regularize local bond and angle geometry. Each model was optimized with the Variable Target Function Method (VTFM) with conjugate gradients (CG). Refinement and energy minimization were achieved by using Amber force field. Among the above models, the most acceptable models were finalized with best fit data to Ramachandran plot. The final 3D models were verified by using the Structural Analysis and Verification Server (SAVES) which has various programs such as PROCHECK, WHAT CHECK and VERIFY 3D (http://nihserver.mbi.ucla.edu/SAVES). The final verified best homology models and experimental structures were further used for the active site binding mode analysis of ASADHs with substrate bAP/ASA. Sequence similarity based clustering was performed using the program CD-HIT[28] (Cluster Database at High Identity with Tolerance) with the 40 % sequence identity filter for the entire primary protein sequences of ASADHs (detail information is given in Table 1). After clustering of sequences, multiple sequence alignment was performed for each cluster with default parameters using the program 378

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ClustalX.[29] The highly conserved regions and important active site binding residues were identified using sequence alignment. The protein folds and structural topology are more conserved in comparison to sequence homology. Hence, the structural comparative studies were also performed using superimposition of experimentally determined 3D (X-Ray crystal structures) as well as the modeled protein structures. Usually, active site residues/structures are highly conserved among distantly related enzymes. The catalytic activity of an enzyme is performed by a small, highly conserved constellation of residues within the active site. The sequence and structural analysis of active site, consensus regions an N-terminal GxxGxVG and a C-terminal GAA sequences were monitored for all clusters of ASADH sequences. Superimposed with the active site known template to obtain active site information for the modeled structure and conserved active site residues were also verified manually in multiple-aligned-sequences. The docking studies were performed by the program AutoDock 4.2.[30] The bAP/ASA molecules were built and minimized with Tripos force field, gasteiger huckel charges using the molecular modeling package SYBYL 7.1.[31] All protein structures and ligands were prepared for docking studies using various parameters such as: polar hydrogen atoms added, non-polar hydrogen atoms merged and defining the rotatable bonds for each ligand. Finally, Kollman united atom charge and atom type parameter were added. The Grid parameters (42 42 42 ) for all proteins were set in such a way that it includes active site. The Lamarckian genetic search algorithm was employed and docking run was set to 20. All other parameters were set to default values such as maximum number of energy evaluation was 2 500 000 per run and maximum number of generations in the genetic algorithm were increased to 270 000.

3 Results and Discussion The ASADHs protein sequences and 3D structures were taken from twelve different infectious microorganism species for comparative structural function and active site binding pattern characterization with their known substrate. In this study, ASADHs protein sequences from a variety of organism were clustered on the basis of 40 % protein sequence identity, and in result of CD-HIT, three clusters were obtained. Cluster 1 has 67 % sequences identity with six ASADHs species: vcASADH1, ecASADH, hiASADH, nmASADH, paASADH, stASADH. Cluster 2 has shown 40 % sequence identity with other six species: spASADH, vcASADH2, bsASADH, cjASADH, hpASADH, mtASADH. Cluster 3 has only one caASADH, because it has shown less than 40 % sequence identity with other ASADHs taken species. Clustering at 30 % sequence identity was also performed and it was observed that caASADH is included in cluster 2. Detailed information of ASADHs about 3D structures, sequence identity and number of species in each


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Characterization of Aspartate b-Semialdehyde Dehydrogenase Family

Figure 1. Sequence alignment among representative members of ASADH family (only conserved region are shown). The important residues from active site point of view are highlighted with rectangles (a). The consensus sequences, highlighted with rectangles (b) are N-terminal GxxGxVG Rossmann fold motif, C-terminal GAA and SGxG sequences.

cluster are shown in Table 1. The multiple sequence alignment of the ASADH enzyme family, which provides the consensus regions an N-terminal GxxGxVG Rossmann fold motif, a C-terminal GAA, SGxG sequences and also aided in positioning the conserved active site residues (Figure 1; all residues numbering of ASADHs are according to Uniprot sequence with their Accession no. as given in Table 1). These consensus regions are also reported in Methanococcus jannaschii (mjASADH).[15] Cluster 1 ASADHs protein se-

quences showed high sequence similarity compared to other clustered protein sequence. Active sites residues of modeled ASADHs were constituted by highly conserved amino acid residues among ASADH family. The ASADH enzymes family can be subdivided into three branches, comprising the enzymes from Gram-negative, Gram-positive bacteria and fungi/archaea. On the basis of sequence similarity, the vcASADH2 (Gram-negative bacteria) and caASADH (fungus) were found to be highly homologous to the

Table 1. Comparative analysis of ASADH enzyme taken from different microorganisms and H-bonding interactions found during docking studies of natural substrate b-aspartyl phosphate Cluster Accession Enzymes No. No. [a] form

Sequence Sequence length identity (%)

PDB IDs[b]

H-bonding with b-aspartyl phosphate

1

371

Arg103, Cys136, Glu243, Lys246, Arg270, His277 Arg101, Asn133, Cys134, Glu240, Lys243, Arg267, His274 Arg102, Asn134, Cys135, Glu241, Lys244, Arg267 Arg102, Asn134, Cys135, Glu241, Arg268, His275 Arg102, Glu241, Lys244, His275

2

3

P44801

hiASADH

Q9KQG2

vcASADH1 370

68

1NWC, 1NWH, 1NX6, 1OZA, 1PQP, 1PQU, 1PR3, 1PS8, 1PU2, 1Q2X, 1TA4, 1TB4 1MB4, 1 MC4, 3PZR, 3Q0E

P0A9Q9

ecASADH

367

72

1BRM, 1GL3, 1T4B, 1T4D

P57008

nmASADH 371

67

Q51344

paASADH. 370

67

P0A1F9

stASADH

70

Q97R26

spASADH 358

*

Modeled (Template used: 1MB4) Modeled (Template used: 1MB4) Modeled (Template used: 1GL3) 2GYY, 2GZ1, 2GZ2, 2GZ3

P23247

vcASADH2 337

46

2QZ9, 2R00

Q04797

bsASADH

346

57

Q59291

cjASADH

343

43

O25801

hpASADH 346

46

P0A542

mtASADH 345

40

Modeled (Template used: 2GZ1) Modeled (Template used: 2GZ1) Modeled (Template used: 2GZ1) 3TZ6, 3VOS

Q5ALM0

caASADH 365

*

3HSK

368

*

Arg103, Asn135, Cys136, Glu242, Lys245, Arg268, His275 Arg99, Asn127, Cys128, Glu220, Lys223, Arg245, His252 Arg101, Asn131, Gln159, Glu213, Lys216, Arg238 Arg101, Asn129, Cys130, Glu218, Lys221, Arg243 Arg103, Asn133, Cys134, Glu217, Arg241 Arg101, Cys131, Gln158, His253 Arg99, Asn129, Cys130, Glu224, Lys227, Arg249, His256 Arg112, Glu211, Arg249

[a] All residues numbering of ASADHs are according to Uniprot sequence with their Accession no. [b] Bold PDB IDs were used for docking studies

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Gram-positive bacteria as compared to Gram-negative bacteria. The ASADH protein sequence homologies in a variety of organism were reported from less than 10 % to as high as 95 % sequence identity.[1] Homology modeling of nmASADH, paASADH, stASADH, bsASADH, cjASADH and hpASADH was performed, using highly identical templates, for which crystal structures of ASADH are reported in PDB. The 3D crystal structure with PDB IDs 1 MB4 for nmASADH (66 %) and paASADH (69 %), 1GL3 for stASADH (96 %) and 2GZ1 for bsASADH (58 %), cjASADH (43 %) and hpASADH (48 %) sequence identity were selected as a template for homology modeling. All 3D molecular models of the ASADHs were built including the coenzymes NADP. Molecular modeling and energy minimizations were performed using the programs Modeller9v7 and UCSF Chimera,[32] respectively. The modeled structures were also verified and validated on SAVES. The Ramachandran plots for the quality of developed models were obtained, and developed models were best fit to Ramachandran plot. Energy minimization of 3D structure is vital for providing the maximum stability to the protein. The final optimized and validated models were used for further docking studies. The overall sequence diversity was found during comparative sequence analysis, while the core active site functional group has been preserved throughout of ASADH family. Although there is a low level of sequence identity between the clusters. However, these enzymes retain the same overall fold and appear to have the same catalytic mechanism. Further, all these modeled structures were superimposed with the templates, hence have high structural similarity (Figure 2), only the loop region showed considerable changes. The consensus regions are an N-terminal

GxxGxVG, C-terminal sequences GAA and SGxG (Figure 1). The amino terminal coenzymes binding domain which resembles a classical Rossmann fold motif, and the carboxyl terminal catalytic and dimerization domain where as a characteristics feature of ASADH family.[15] The active site region showed the same structural topology while the NADP binding region showed structural differences between cluster 1 and cluster 2. In case of cluster 1, the NADP binding site has large and more flexible coenzymes binding loop, which is conserved (Figure 2a). While in the cluster 2, the NADP binding site has same number of amino acid residues but the NADP binding pocket is highly constrained and less conserved (Figure 2b). The differences among the various ASADHs at dimer interface surface area are correlated with their catalytic activity. The substantial difference in a-helix sub domain (deletion of residues), which also form the top portion of dimer interface were reported in vcASADH2.[33] These significant differences were also observed in the ASADHs of cluster 2 and caASADH. The molecular docking studies provide valuable information about the residues involved in enzyme-substrate interaction. For such interaction studies, the most important requirement was the proper orientation and conformation of substrate (bAP/ASA in this case) which fitted to the enzyme (ASADH family) active site appropriately and formed enzyme-substrate intermediate complex. Therefore, optimal interactions and best AutoDock score were used as criteria to interpret the best pose amongst the 20 conformations generated by the program AutoDock. The docking parameterization and protocol were validated using both known docked conformation of co-crystal ligand and superimposition of docked conformation with

Figure 2. The overall structural superimposition of modeled and 3D crystal structures of ASADH family. (a) Cluster 1, showing the NADP binding site has a large and more flexible coenzymes binding loop which is conserved. (b) Cluster 2, the NADP binding site has the same number of amino acids residues in this region but is less conserved and highly constrained NADP binding pocket

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Figure 3. Best docked conformation of bAP and ASA into the active site of ASADHs. (a) ecADADH: superimposition of docked conformation of bAP (white), ASA (magenta) and co-crystallized AsO4 (PDB ID:1T4B) (red); (b) mtADADH: (PDB ID: 1TZ6) superimposition of docked conformation of bAP (white), ASA (magenta) and co-crystallized SMCS (gray) and SO4 group (yellow).

co-crystal conformation of ligand (Figure 3). The best docked confirmation of bAP/ASA molecule reveled that carboxylic and phosphate group fitted the active site in a perfect manner. The docking studies were perfectly in agreement with earlier published results.[26,34] Interaction studies of this substrate observed the presence of active site residues Arg102, Asn134, Cys135, Glu241, Lys244, Arg267 and His274 in ecASADH, and Arg99, Asn129, Cys130, Glu224, Lys227, Arg249 and His256 in mtASADH and others also

(Figure 4–5). The docked conformation of phosphate group is superimposed with the AsO4 (Arsenate, co-crystal inhibitor in PDB ID: 1TA4) as depicted in Figure 3a, and residues Lys244 NZ, Arg102 NH1 and NH2 interacted with O atom of Phosphate in ecASADH (Figure 4c), similar type of interactions for AsO4 were also reported earlier.[35] In mtASADH, phosphate group also located near the SO4 and both carboxylic groups of bAP/ASA (docked conformation) and SMCS (S-methyl-l-cysteine sulfoxide; structure with cova-

Figure 4. Binding interactions of substrate in the active site of ASADHs (Cluster 1) obtained by docking studies. (a) hiASADH; (b) vcASADH; (c) ecASADH; (d) nmASADH; (e) paASADH; (f) stASADH. The dotted line showed the H-binding interactions

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Figure 5. Binding interactions of substrate in the active site of ASADHs (Cluster 2) obtained by docking studies. (a) spASADH; (b) vcASADH2; (c) bsASADH; (d) cjASADH; (e) hpASADH; (f) mtASADH. The dotted line showed the H-binding interactions

lent bounded SMCS inhibitor and SO4 in PDB ID: 3TZ6[36]) are engaged in a bidentate interaction with the guanidinium N atom of Arg249 and the NE2 atom of His256 (Figure 3b & 5f). The overall binding interaction of bAP/ASA is similar to SMCS binding interactions. The various forms of ASADH were used for this validated molecular docking studies, and for the comparative analysis of their active site binding mode. Both functional carboxylic and phosphate group of substrate interact with two key arginyl active site residues of ASADHs. The catalytic residues cysteine, which is covalent linked to the intermediate in the reaction while histidine act as an acid/base catalyst.[37] The active site residues histidine (residues no. of ASADHs have been given in Table 1, and Figures 4 and 5) also formed the H-bonding with the carboxylic group of substrate. Best docked conformation of the carbonyl and ammonium group of substrate interacted with cysteine and side chain carboxylic functional group of glutamine acid (numbering of residues mentioned in Table 1 and Figures 4 and 5), respectively. The details of H-bonding between substrate and active site residues of ASADHs are shown in Figures 4 and 5. These valuable information will further support the computational drug designing and inhibitor reorganization.

ing the mode of action, in silico insight into the 3D structure, enzyme-substrate interactions with natural substrate in this homologous enzyme family. The active site residues are conserved among this family. The catalytic residue cysteine, which is covalently linked to the intermediate in the reaction while histidine act as an acid/base catalyst. During comparative sequence analysis, high diversity was found in the sequences of different ASADHs and exhibited similar binding interactions with the substrate. Both functional carboxylic and phosphate group of substrate showed interactions with the guanidinium N atom of two key arginyl active site residues of ASADHs. These structural and active site binding mode characterizations will provide the basis for designing the more potent and selective substrate analogues inhibitors against ASADH family. Moreover, these studies can also be helpful in development of novel inhibitors against various pathological microbes species using ligand and structural based drug design approaches.

Acknowledgements 4 Conclusions In this work, molecular modeling and comparative active site binding mode studies were performed for understand382

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Rajender Kumar acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing Senior Research Fellowship [Grant File No. 09/727(0100)/ 2012-EMR-I].


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Received: October 23, 2012 Accepted: March 19, 2012 Published online: April 9, 2013

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