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Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study a
b
Ankur Chaudhuri , Indrani Sarkar & Sibani Chakraborty
a
a
Department of Microbiology, West Bengal State University, Barasat, Berunanpukuria, P.O. Malikapur, North 24 Parganas, Kolkata 700126, West Bengal, India b
Department of Physics, Narula Institute of Technology, 81, Nilgunj Road, Agarpara, Kolkata 700109, West Bengal, India Published online: 27 Nov 2013.
To cite this article: Ankur Chaudhuri, Indrani Sarkar & Sibani Chakraborty (2014) Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study, Journal of Biomolecular Structure and Dynamics, 32:12, 1969-1978, DOI: 10.1080/07391102.2013.848173 To link to this article: http://dx.doi.org/10.1080/07391102.2013.848173
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Journal of Biomolecular Structure and Dynamics, 2014 Vol. 32, No. 12, 1969–1978, http://dx.doi.org/10.1080/07391102.2013.848173
Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study Ankur Chaudhuria, Indrani Sarkarb and Sibani Chakrabortya* a
Department of Microbiology, West Bengal State University, Barasat, Berunanpukuria, P.O. Malikapur, North 24 Parganas, Kolkata 700126, West Bengal, India; bDepartment of Physics, Narula Institute of Technology, 81, Nilgunj Road, Agarpara, Kolkata 700109, West Bengal, India Communicated by Ramaswamy H.Sarma
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(Received 5 July 2013; accepted 20 September 2013) Meprins are complex and highly glycosylated multi-domain enzymes that require post-translational modifications to reach full activity. Meprins are metalloproteases of the astacin family characterized by a conserved zinc-binding motif (HExxHxxGFxHExxRxDR). Human meprin-α and -β protease subunits are 55% identical at the amino acid level, however the substrate and peptide bond specificities vary markedly. Current work focuses on the critical amino acid residues in the non-primed subsites of human meprins-α and -β involved in inhibitor/ligand binding. To compare the molecular events underlying ligand affinity, homology modeling of the protease domain of humep-α and -β based on the astacin crystal structure followed by energy minimization and molecular dynamics simulation of fully solvated proteases with inhibitor Pro-Leu-Gly-hydroxamate in S subsites were performed. The solvent accessible surface area curve shows a decrease in solvent accessibility values at specific residues upon inhibitor binding. The potential energy, total energy, H-bond interactions, root mean square deviation and root mean square fluctuation plot reflect the subtle differences in the S subsite of the enzymes which interact with different residues at P site of the inhibitor. Keywords: α- and β-meprins; hydroxamic acid derivative; solvent accessibility; simulation; subsites
1. Introduction Astacin, a zinc endopeptidase from the crayfish Astacus astacus L (Stocker, Wolz, Zwilling, Strydom, & Auld, 1988; Titani et al., 1987) represents a structurally distinct group of metalloproteases termed as astacin family. They have been found to play a diverse role in both mature and developmental systems ranging from bone morphogenesis, tissue differentiation, hatching process, digestive function, and in different diseases including cancer (Bond & Beynon, 1995; Dumermuth et al., 1991; Kessler, Takahara, Biniamov, Brusel, & Greenspan, 1996; Stocker, Gomis-Ruth, Bode, & Zwilling, 1993). In humans six astacins are known, namely meprin-α and -β (Bond & Beynon, 1995), bone morphogenic protein-1 (BMP-1) with its major splice variant mammalian tolloid, mammalian tolloid-like enzymes (Hopkins, Keles, & Greenspan, 2007), and ovastacin (Quesada, Sanchez, Alvarez, & Lopez-Otin, 2004). Meprins are the only astacin proteases that function on the membrane extracellularly because they can be membrane bound or secreted. Only recently it has become evident that meprins exhibit a much broader expression pattern, implicating functions in angiogenesis, cancer, inflammation, fibrosis, and neurodegenerative diseases (Broder & Becker-Pauly, 2013). The catalytic protease domain of *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
meprin-α and -β comprises about 198 amino acid residues and shares a sequence identity of 30 and 37%, respectively to astacin, the three-dimensional structure of which is known (PDB 1AST at 1.8 Å resolution) (Bode, Gomis-Rüth, Huber, Zwilling, & Stöcker, 1992). Meprins of the astacin family have an extended sequence HExxHxxGFxHExxRxDR containing the Zn-binding motif HEXXH which is characteristic of all metalloendopeptidases (Rawling & Barrett, 1995). In the absence of experimental structures, computational methods are used to predict three-dimensional protein models to provide insight into the structure and function of proteins. A knowledge based homology model of the protease domain of human meprins-α and -β have been done using the template astacin (PDB ID: 1AST). The active site cleft is subdivided into an N-terminal or upstream region with respect to the scissile bond, the ‘non-primed’ side, and a C-terminal or downstream region, the ‘primed’ side. Consequently, the substrate/inhibitor side chains on the non-primed side away from the scissile bond are termed P1, P2, P3, etc. and their cognate enzyme subsites S1, S2, S3, etc. On the primed side, substrate/inhibitor side chains P1′, P2′, P3′, etc. are accommodated in subsites S1′, S2′, S3′, etc (Abramowitz, Schechter, & Berger, 1967; Schechter & Berger,
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1967). The catalytic domain is stabilized by two intradomain disulphide bridges and several conserved salt bridges. Multiple sequence alignment analysis indicated that 50 amino acids including three cysteine residues are strictly conserved. Human meprins are inhibited by hydroxamic acid derivatives such as Pro-Leu-Gly-hydroxamate which is also a therapeutic target for matrix metalloproteinases (MMPs) (Broder & Becker-Pauly, 2013). It has been shown by modeling that the hydroxamate-based inhibitor, Pro-Leu-Gly-hydroxamate, binds to the non-primed sites of human meprin-α and -β. The catalytic zinc is penta-coordinated with three histidine residues of human meprins (αHis90/βHis91, αHis94/ βHis95, and αHis100/βHis101), carbonyl oxygen of glycine, and ‘O’ atom of hydroxamate of the inhibitor in the complex. An important anchoring function in this interaction is played by the niche-like S3 subsite of the β-edge strand, which harbors the inhibitor’s proline ring (Bode et al., 1994; Grams et al., 1996). The hydroxamic acid derivative inhibitor modeled in the S1, S2, and S3 subsites of the active site cleft of human meprin-α and -β structure has been refined by explicit solvent MD simulations. Analysis of the MD refined complexes help in identifying the different interactions contributing to protein stability and structural flexibility at protein-inhibitor binding sites. The different residues at the P1, P2, and P3 positions of the inhibitor interacting with different amino acid residues in S1, S2, and S3 subsites of human meprin-α and -β helps in determining the substrate specificity of the two enzymes. 2. Materials and methods 2.1. Homology modeling and docking The structures of protease domain of human meprin-α and -β were first modeled using the X-ray crystal structure of astacin (1AST) at 1.8 Å resolution as the template. Multiple sequence alignment was performed with ClustalW (Thompson, Higgins, & Gibson, 1994) using a gap penalty of 10 and employing the GONNET matrix (Larkin et al., 2007) to know the conserved residues among the sequences. Sequence homology (above 30% identity) of the meprins with astacin was used to construct the homology models using Discovery Studio 3.5 (Accelrys Inc., San Diego, CA) based on MODELLER 9v10 (MartiRenom et al., 2000; Sali & Blundell, 1993). From the resultant 100 models generated, the best model was selected on the basis of lowest probability density function (PDF) score. The generated modeled protein was checked to correct any improper bond lengths, bond angles, conformations as well as add missing atoms and correct connectivity within the protein. The stereochemical quality of the models was validated by Ramachandran plot using PROCHECK (Laskowski, MacArthur, Moss, & Thornton, 1993; Morris, MacArthur, Hutchinson, & Thornton,
1992). Docking was done by superimposing the modeled meprins on the crystal structure of astacin complexed with Pro-Leu-Gly-hydroxamate (PDB ID:1QJJ). Solvent accessible surface area (SASA) can be used to predict the binding – induced conformational changes of the protein – inhibitor complexes. It was calculated by setting a probe radius of 1.40 Å and 240 grid points per atom using Discovery Studio 3.5. Two models of the protease domain of human meprin-α and -β in complex with Pro-Leu-Gly-hydroxamate inhibitor was used for MD simulations. 2.2. Molecular dynamic simulations All MD simulations were performed using Discovery Studio 3.5 with the CHARMm forcefield (Feller & Mackerell, 2000; Mackerell et al., 1998). Both models of human meprin-α and -β with hydroxamic acid derivative inhibitor was solvated with the TIP3P water molecules (Jorgensen, Chandrasekhar, Madura, Impey, & Klein, 1983) in an explicit spherical boundary condition (Brunger, Brooks, & Karplus, 1984) with a minimum distance of 7 Å from boundary to the protein atom. Each simulation was carried out by Standard Dynamics Cascade protocol of Discovery Studio 3.5, which includes two stages of minimization followed by heating, equilibrium, and production run. Positional constraints were imposed on the NE2 atom of three His residues at the active site with the zinc atom to avoid any drastic rearrangement of these residues. A distance cut-off of 10 Å from the inhibitor was used to select all atoms within that region encircling the inhibitor-bound active site of the protease domain on which dynamic simulation was run. All atoms outside the 10 Å region was fixed applying harmonic restraint. The solvated protease inhibitor models were first minimized using 500 steps of steepest descent followed by 1000 steps of conjugate gradient method to remove steric clashes and to better soak the water molecules into the macromolecules. This was done to remove any unfavourable contacts present in the models. The minimized structures were then subjected to heating from 50 to 300 K for 120 ps and equilibrated for 1 ns. Long-range electrostatic interactions were treated with a spherical cut-off method. Non-bond higher and lower cut-off distance were maintained at 12 Å and 10 Å respectively. Both complexes were further simulated for 20 ns at 300 K with a time step of 2 fs for production run. This was carried in the NVT ensemble. During the simulations, all covalent bonds involving hydrogen were constrained using the SHAKE algorithm (Ryckaert, Ciccotti, & Berendsen, 1977). The trajectories were saved every 50 ps interval for analysis. 2.3. Data evaluation The SASA was calculated to measure the differences of solvent accessibility of residues before and after inhibitor
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Comparative analysis of binding sites of human meprins binding. The root-mean square deviation (RMSD) of the protein backbone atoms from the initial coordinates was used as a measure for structural differences. The rootmean square fluctuation (RMSF) of the backbone atoms relative to the average structure over the last 5 ns simulations was calculated as an indicator of structural flexibility. RMSF gives a measure of the relative standard deviation for each residue in human meprins-α and -β. To find out the hydrogen bond stability of the protein-inhibitor complexes throughout the simulation period, we performed the heat map protocol (Dokla et al., 2012). Analysis of salt bridge was done using an oxygen–nitrogen distance cut-off of 5.0 Å between charged residue side chains. All the above analyses were performed with Accelrys Discovery Studio 3.5 (Accelrys, v 3.5.0.12158). 3. Results and discussion 3.1. Multiple sequence analysis Multiple sequence alignment of the protease domain of human meprins-α and -β (humep-α and humep-β) share a sequence identity of 30 and 37%, respectively, to astacin (Figure 1). Sequence alignment indicates that 50 residues including three cysteine are strictly conserved. Homology models built using the three-dimensional structure of astacin (PDB: 1AST) reveal structural similarity among the proteases. The models were sorted according to lowest PDF scores. Validation of the models show above 90% residues in allowed region of Ramachandran plot. Superposition of humep-α and -β models on the template astacin structure (1AST) show C–α RMSD values of 1.41 Å and 0.67 Å, respectively. 3.2. Protein – ligand binding We analyzed the intermolecular interactions of generated inhibitor docked structure of both human meprin complexes (Table 1). In case of humep-α complex, six
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intermolecular hydrogen bonds involving residues Cys63, Ser65, Glu66, and Tyr149 were detected. Apart from six H-bonds, electrostatic interactions involving two salt bridges between Glu66 of the protease and Proline residue of inhibitor were observed. In case of humep-β complex, five intermolecular hydrogen bonds involving residues Cys63, Ser65, Glu92, and Tyr150 were detected. Apart from these, one pi-cation interaction between Trp64 of the protease and Pro residue of inhibitor was also observed. In both human meprin complexes residues Cys63–Val67 forms the upper edge of the active site cleft, so that the carbonyl group of Cys63 presumably plays a role in hydrogen-bonding and polarizing the amino group of hydroxamic acid of bound inhibitor. The hydrogen bonds and electrostatic interactions add stability to the structures (Bella & Humphries, 2005; Xu, Tsai, & Nussinov, 1997). The total SASA is the surface of a biomolecule that is accessible to a solvent. The burial of hydrophobic amino acids in the protein is a driving force in protein folding. The extent to which an amino acid interacts with the solvent and the protein core is naturally proportional to the surface area exposed to these environments (Durham, Dorr, Woetzel, Staritzbichler, & Meiler, 2009). The SASA curve for complexed and non-complexed human meprins-α and -β is represented in Figure 2(a) and (b), and the residues that differ in solvent accessibility upon inhibitor binding are shown in Table 2. The SASA analysis of meprins before and after inhibitor binding reveals that there is decrease in solvent accessibility value for meprin residues interacting with hydroxamic acid derivative inhibitor. From the SASA plot, we observed that His90 that acts as a Zn-binding ligand in penta-coordinated structure of the metalloprotease domain is not involved in inhibitor binding in case of humep-α – inhibitor complex. The interacting residues found from SASA analysis are the ones involved in hydrogen bonding and other electrostatic interactions.
Figure 1. Multiple sequence alignment of protease domain of human meprin-α and -β with crystal structure of astacin (1AST). ‘*’ strands for identical residues, ‘:’ strands for conserved substitutions and ‘.’ strands for semi-conserved substitutions.
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Table 1.
Intermolecular interaction of docked complexes.
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Sl. No
Bond
Ligand residue
Receptor residue
Interaction constituents
Distance
(a) Humep-α – hydroxamic acid derivative complex 1 H bond1 LEU 2 H bond2 GLY 3 H bond3 HOA 4 H bond4 PRO 5 H bond5 HOA 6 H bond6 HOA 7 Salt bridge1 PRO 8 Salt bridge2 PRO
SER65 TYR149 TYR149 GLU66 CYS63 TYR149 GLU66 GLU66
SER65:N – LEU:O TYR149:OH – GLY:O TYR149:OH – HOA:O GLU66:OE1 – PRO:NHT1 CYS63:O – HOA:HN1 TYR149:OH – HOA:HO GLU66:OE1 – PRO:N GLU66:OE2 – PRO:N
2.3512 3.0414 2.364 2.3372 1.7426 2.4179 2.59634 4.31529
(b) Humep-β – hydroxamic acid derivative complex 1 H bond1 LEU 2 H bond2 HOA 3 H bond3 LEU 4 H bond4 HOA 5 H bond5 HOA 6 Pi-cation PRO
SER65 TYR150 SER65 CYS63 GLU92 TRP64
SER65:N – LEU:O TYR150:OH – HOA:O SER65:O – LEU:HN CYS63:O – HOA:HN1 GLU92:OE2 – HOA:HO TRP64 – PRO:N
2.7748 3.0393 1.984 1.817 2.274 5.7778
3.3. Global stability of the system Stability of the system was evaluated in terms of potential energy (PE) and total energy (TE) as a function of simulation time. Both the energy profiles are lower in case of humep-β complex and remained constant throughout 20 ns production run in both human meprin complexes (Figure 3(a) and (b)). 3.4. Protein structural features The meprin-hydroxamic acid stability in the 20 ns simulation was verified by monitoring the time evolution of structural parameters. The main chain RMSDs of modeled humep-α and humep-β – hydroxamic acid complexes (Figure 4) with respect to the initial structures as a function of time throughout 20 ns molecular dynamics simulation were analyzed to find deviations at the structural level. The production phase of dynamic simulation started at 1.12 ns after completing 120 ps of heating and 1 ns of equilibration. The results reveal that the RMSD plot of both humep complexes increased from the initial phase of the production run to 0.26 nm which remained more or less stable around 0.25–0.27 nm during the production run. For humep-β complex, the RMSD plot remained stable for the remaining period of the production analysis. In case of humep-α complex, the RMSD value increases and fluctuates with a larger amplitude of 0.29 nm at ~15.12 ns and remained stable at that value for the remaining 6.12 ns simulation period. Inhibitor binding is related to protein flexibility at the protein-ligand binding site. To understand the conformational flexibility of the protein-inhibitor complex at different binding site residues we evaluated the RMSF of the main chain as a function of residue number. Figure 5(a) and (b) show the specific residues of humep-α
and humep-β involved in Pro-Leu-Gly-hydroxamate binding. The results of the RMSF plot indicate that for humep-α – hydroxamic acid complex there were significant fluctuations around residues 60–69, 70–80, 86–95, 97–103, 121–127, 129–131, and 146–149, respectively. The inhibitor bound complex of humep-β exhibited fluctuations at residues 59–70, 73–81, 86–96, 98–105, 122–129, 131–133, 146–151, and 171–174, respectively. Residues α/βCys63, α/βTrp64, and αAsn125/βAsn126 comprise the S1 subsite of human meprins. S2 subsite consists of residues α/βSer65, αTyr99/βTrp100, and αAsp131/ βAsp132, and S3 subsite contains residues α/βTrp64 and αGlu66/βSer66. RMSF curves clearly indicate strong fluctuations in these non-primed subsite regions of the metalloproteases involved in inhibitor binding. A higher structural flexibility is observed at residue Gly68 having greater amplitude for humep-α complex (~0.25 nm) compared to humep-β complex (~0.15 nm). However, residues 63 and 64 comprising both S1 and S3 subsite region show same structural fluctuations for both complexes. Ser65 which is one of the S2 subsite residues, show greater fluctuations for meprin-α than meprin-β complex. In humep-α complex, greater fluctuations at residue Ser65 and Gly68 may be due to the presence of negatively charged amino acids, Glu66 and Asp69 adjacent to the respective amino acids, whereas the corresponding amino acid residues in humep-β complex are Ser66 and Asn69. In humep-α, Glu66 that forms the salt bridges with Proline residue of the inhibitor have greater fluctuation than corresponding Ser66 of humep-β complex. Greater structural flexibility at residues 146–149 is observed for humep-β complex (~0.18 nm) than humep-α complex (~0.12 nm). Additional fluctuation at residues 171–174 is reported for humep-β which is not found in humep-α complex
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Comparative analysis of binding sites of human meprins
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Figure 2. SASA plot of human meprin-α (a) and -β (b) before and after docking. Before docking is represented with blue line and after docking is represented with red line in the plot. Table 2.
Residues of human meprins that differ in solvent accessibility upon inhibitor binding.
Protein
Residues name
Humep-α-HA Humep-β-HA
Cys63, Trp64, Ser65, Glu66, Val67, Asn75, Glu91, His94, Tyr99, His100, His124, Tyr149 Cys63, Trp64, Ser65, Ser66, Val67, Arg71, His91, Glu92, His95, Trp100, His101, Tyr150
3.5. Hydrogen bonding In order to characterize the differences in conformational dynamics of the meprin-hydroxamic acid derivative complexes, we examined the hydrogen-bonding pattern in the 20 ns simulations. The stability of the
protein – ligand complex can be explored in terms of hydrogen bonding, hence we focused on the behavior of the hydrogen bonds present at the binding sites of the protein – inhibitor complexes. Majority of the hydrogen bonds involve main chain-side chain O⋯H–O
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Figure 3.
Stability of the system. (a). PE vs. Time (b). TE vs. Time.
Figure 4. Comparison of trajectory of the root-mean square deviation of human meprin-α and -β complexes with respect to its initial structure. Blue and Red line indicates humep-α and -β respectively.
and O⋯H–N hydrogen bonds. The hydrogen bond interactions of the last 5 ns average structure were obtained from MD simulations of both human meprin complexes (Table 3). It shows that some of the H-bonds present in the modeled structures of both meprin-inhibitor
complexes before simulation were lost and some new H-bonds were generated on completion of the 20 ns production run. Analysis of the stability of the H-bonds from last 5 ns average structure in both cases revealed that the H-bonds Glu66:OE1 – Pro:NHT1 and
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Comparative analysis of binding sites of human meprins
Figure 5.
Table 3. Sl. No
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Root-mean square fluctuations per residue of human meprin-α (a) and -β (b) hydroxamic acid derivative complexes. H-bond interaction of last 5 ns average structure. Bond
Ligand Residue
(a) humep-α – hydroxamic acid derivative 1 H bond1 LEU 2 H bond2 PRO 3 H bond3 LEU (b) humep-β – hydroxamic acid derivative 1 H bond1 LEU 2 H bond2 HOA 3 H bond3 HOA
Receptor Residue
Interaction Constituents
Distance
SER65 GLU66 SER65
SER65:HN – LEU:O GLU66:O – PRO:NHT1 SER65:O – LEU:HN
2.1534 1.7655 2.076
SER65 CYS63 GLU92
SER65:O – LEU:HN CYS63:O – HOA:HN1 GLU92:OE2 – HOA:HO
2.0334 2.3405 1.9556
Ser65:O – Leu:HN were present throughout the 20 ns simulation in case of humep-α complex. In case of humep-β complex, the two H-bonds Ser65:O – Leu:HN and Glu92:OE2 – HOA:HO persist throughout simulation. The Ser65 residue present at S2 subsite is involved in main chain – main chain hydrogen bonding for both the humep complexes. S3 subsite residue Glu66 of humep-α complex is involved in main chain – side chain hydrogen bonding with Proline of hydroxamic acid derivative. However, identical residue Ser66 present in the S3 subsite region of humep-β complex is not involved in hydrogen bonding with the inhibitor.
This indicates that P3 site of inhibitor has a preference for acidic residue at S3 subsite to form H-bond imparting stability to protein inhibitor complex. Hydrogen bond involving tyrosine residue present in the SXMHY domain (Met-turn) of the metalloproteases is observed before simulation but absent after simulation in both the cases. The last 5 ns average structure of both human meprin-inhibitor complexes show that the carbonyl oxygen of glycine and oxygen atom of hydroxamic acid of the inhibitor together with the three active site histidine residues of meprins maintain the penta-covalent geometry of the active site cleft (Figure 6).
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Figure 6. Hydrogen bonding network and Zn co-ordination of human meprin-α (a) and -β (b). Inhibitor shown in ball and stick and important residues present in active site shown in stick. Inhibitor coordinates the catalytic zinc ion (grey sphere) via carbonyl oxygen of glycine and ‘O’ atom of hydroxamic acid. Figure created with PyMol (www.pymol.org).
4. Conclusion In this article, we performed MD simulation to compare and analyze the substrate/inhibitor binding sites of human meprins-α and -β. An inhibitor Pro-Leu-Gly-hydroxamate was modeled into the active site cleft of both meprins following the mode of binding of the inhibitor in the active site of astacin. It is to be mentioned that studies on conformational flexibility as shown by RMSF calculation point out certain flexible regions in the
meprin structures, which may have an important role in imparting stability to the meprin-hydroxamic acid derivative complex. Analysis of simulation trajectories indicated that some H-bonds involving S1, S2, and S3 subsite residues of meprins Glu66:OE1 – Pro:NHT1, Ser65:O – Leu:HN, and Ser65:O – Leu:HN, Glu92:OE2 – HOA:HO, persist throughout the simulation period in humep-α and -β complexes, respectively. S2 subsite residue Ser65 present in both human meprin complexes
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Comparative analysis of binding sites of human meprins may have an important role in ligand binding as reflected from H-bonding analysis. Human meprin α and β have distinct specificities for preferred amino acids at or near the cleavage site due to subtle changes in amino acids in the corresponding S1, S2, and S3 subsites of the active site cleft which in turn to some extent explains the catalytic activity of the proteases. This knowledge is required for the structural alteration in the inhibition of some of these ligands in order to modify the biological properties of this protein. Further studies with other ligands binding to both primed and non-primed region of meprins could give more insight into the mode of action of meprin inhibitors. The information of receptor-ligand interaction is important and it guides the selections of candidature sites for further experimental data studies. List of Abbreviations HOA hydroxamic acid humep-α – HA human meprin-α – Pro-Leu-Gly-hydroxamate humep-β – HA human meprin-β – Pro-Leu-Gly-hydroxamate
Acknowledgement Our sincere thanks to Dr Asim Bera for his assistance in the preparation of Figure 6 using Pymol.
Funding We are thankful to the Department of Biotechnology (DBT), Government of India for their financial support.
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Dumermuth, E., Sterchi, E. E., Jiang, W. P, Wolz, R. L., Bond, J. S., Flannery, A. V., & Beynon, R. J. (1991). The astacin family of metalloendopeptidases. Journal of Biological Chemistry, 266, 21381–21385. Durham, E., Dorr, B., Woetzel, N., Staritzbichler, R., & Meiler, J. (2009). Solvent accessible surface area approximations for rapid and accurate protein structure prediction. Journal of Molecular Modeling, 15, 1093–1108. Eman, M. D., Amr, H. M., Mohamed, S. A. E., Ahmed, H. ElK., Michael, W. L., & Khaled, A. A. (2012). Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors. PLoS ONE, 7, e49284. Feller, S. E., & Mackerell, A. D. (2000). An improved empirical potential energy function for molecular simulations of phospholipids. Journal of Physical Chemistry B, 104, 7510–7515. Grams, F., Dive, V., Yiotakis, A., Yiallouros, I., Vassiliou, S., Zwilling, R., …, Stocker, W. (1996). Structure of astacin with a transition-state analogue inhibitor. Naturel Structural Biology, 3, 671–675. Hopkins, D. R., Keles, S., & Greenspan, D. S. (2007). The bone morphogenetic protein 1/Tolloid-like metalloproteinases. Matrix Biology, 26, 508–523. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., & Klein, M. L. (1983). Comparison of simple potential functions for simulating liquid water. Journal of Chemical Physics, 79, 926–935. Kessler, E., Takahara, K., Biniamov, L., Brusel, M., & Greenspan, D. S. (1996). Bone morphogenetic protein-1: the type I procollagen C-proteinase Science, 271, 360–362. Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., … Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947–2948. Laskowski, R. A., MacArthur, M. W., Moss, D. S., & Thornton, J. M. (1993). Procheck: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283–291. Mackerell, A. D., Bashford, D., Bellott, M., Dunbrack, R. L., Evanseck, J. D. … Karplu, M. (1998). All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. Journal of Physical Chemistry B, 102, 3586–3616. Marti-Renom, M. A., Stuart, A., Fiser, A., Sanchez, R., Melo, F., & A., Sali (2000). Comparative protein structure modeling of genes and genomes. Annual Review of Biophysics and Biomolecular Structure, 29, 291–325. Morris, A. L., MacArthur, M. W., Hutchinson, E. G., & Thornton, J. M. (1992). Stereochemical quality of protein structure coordinates. Proteins: Structure, Function and Genetics, 12, 345–364. Quesada, V., Sanchez, L. M., Alvarez, J., & Lopez-Otin, C. (2004). Identification and characterization of human and mouse ovastacin: A novel metalloproteinase similar to hatching enzymes from arthropods, birds, amphibians, and fish. Journal of Biological Chemistry, 279, 26627–26634. Rawling, N. D., & Barrett, A. J. (1995). Evolutionary families of metallopeptidases. Methods in Enzymology, 248, 183–228. Ryckaert, J. P., Ciccotti, G., & Berendsen, H. J. C. (1977). Numerical integration of the Cartesian Equations of Motion of a System with Constraints. Journal of Computational Physics, 23, 327–341. Sali, A., & Blundell, T. L. (1993). Comparative protein modelling by satisfaction of spatial restraints. Journal of Molecular Biology, 234, 779–815.
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study a
b
Ankur Chaudhuri , Indrani Sarkar & Sibani Chakraborty
a
a
Department of Microbiology, West Bengal State University, Barasat, Berunanpukuria, P.O. Malikapur, North 24 Parganas, Kolkata 700126, West Bengal, India b
Department of Physics, Narula Institute of Technology, 81, Nilgunj Road, Agarpara, Kolkata 700109, West Bengal, India Published online: 27 Nov 2013.
To cite this article: Ankur Chaudhuri, Indrani Sarkar & Sibani Chakraborty (2014) Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study, Journal of Biomolecular Structure and Dynamics, 32:12, 1969-1978, DOI: 10.1080/07391102.2013.848173 To link to this article: http://dx.doi.org/10.1080/07391102.2013.848173
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Journal of Biomolecular Structure and Dynamics, 2014 Vol. 32, No. 12, 1969–1978, http://dx.doi.org/10.1080/07391102.2013.848173
Comparative analysis of binding sites of human meprins with hydroxamic acid derivative by molecular dynamics simulation study Ankur Chaudhuria, Indrani Sarkarb and Sibani Chakrabortya* a
Department of Microbiology, West Bengal State University, Barasat, Berunanpukuria, P.O. Malikapur, North 24 Parganas, Kolkata 700126, West Bengal, India; bDepartment of Physics, Narula Institute of Technology, 81, Nilgunj Road, Agarpara, Kolkata 700109, West Bengal, India Communicated by Ramaswamy H.Sarma
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(Received 5 July 2013; accepted 20 September 2013) Meprins are complex and highly glycosylated multi-domain enzymes that require post-translational modifications to reach full activity. Meprins are metalloproteases of the astacin family characterized by a conserved zinc-binding motif (HExxHxxGFxHExxRxDR). Human meprin-α and -β protease subunits are 55% identical at the amino acid level, however the substrate and peptide bond specificities vary markedly. Current work focuses on the critical amino acid residues in the non-primed subsites of human meprins-α and -β involved in inhibitor/ligand binding. To compare the molecular events underlying ligand affinity, homology modeling of the protease domain of humep-α and -β based on the astacin crystal structure followed by energy minimization and molecular dynamics simulation of fully solvated proteases with inhibitor Pro-Leu-Gly-hydroxamate in S subsites were performed. The solvent accessible surface area curve shows a decrease in solvent accessibility values at specific residues upon inhibitor binding. The potential energy, total energy, H-bond interactions, root mean square deviation and root mean square fluctuation plot reflect the subtle differences in the S subsite of the enzymes which interact with different residues at P site of the inhibitor. Keywords: α- and β-meprins; hydroxamic acid derivative; solvent accessibility; simulation; subsites
1. Introduction Astacin, a zinc endopeptidase from the crayfish Astacus astacus L (Stocker, Wolz, Zwilling, Strydom, & Auld, 1988; Titani et al., 1987) represents a structurally distinct group of metalloproteases termed as astacin family. They have been found to play a diverse role in both mature and developmental systems ranging from bone morphogenesis, tissue differentiation, hatching process, digestive function, and in different diseases including cancer (Bond & Beynon, 1995; Dumermuth et al., 1991; Kessler, Takahara, Biniamov, Brusel, & Greenspan, 1996; Stocker, Gomis-Ruth, Bode, & Zwilling, 1993). In humans six astacins are known, namely meprin-α and -β (Bond & Beynon, 1995), bone morphogenic protein-1 (BMP-1) with its major splice variant mammalian tolloid, mammalian tolloid-like enzymes (Hopkins, Keles, & Greenspan, 2007), and ovastacin (Quesada, Sanchez, Alvarez, & Lopez-Otin, 2004). Meprins are the only astacin proteases that function on the membrane extracellularly because they can be membrane bound or secreted. Only recently it has become evident that meprins exhibit a much broader expression pattern, implicating functions in angiogenesis, cancer, inflammation, fibrosis, and neurodegenerative diseases (Broder & Becker-Pauly, 2013). The catalytic protease domain of *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
meprin-α and -β comprises about 198 amino acid residues and shares a sequence identity of 30 and 37%, respectively to astacin, the three-dimensional structure of which is known (PDB 1AST at 1.8 Å resolution) (Bode, Gomis-Rüth, Huber, Zwilling, & Stöcker, 1992). Meprins of the astacin family have an extended sequence HExxHxxGFxHExxRxDR containing the Zn-binding motif HEXXH which is characteristic of all metalloendopeptidases (Rawling & Barrett, 1995). In the absence of experimental structures, computational methods are used to predict three-dimensional protein models to provide insight into the structure and function of proteins. A knowledge based homology model of the protease domain of human meprins-α and -β have been done using the template astacin (PDB ID: 1AST). The active site cleft is subdivided into an N-terminal or upstream region with respect to the scissile bond, the ‘non-primed’ side, and a C-terminal or downstream region, the ‘primed’ side. Consequently, the substrate/inhibitor side chains on the non-primed side away from the scissile bond are termed P1, P2, P3, etc. and their cognate enzyme subsites S1, S2, S3, etc. On the primed side, substrate/inhibitor side chains P1′, P2′, P3′, etc. are accommodated in subsites S1′, S2′, S3′, etc (Abramowitz, Schechter, & Berger, 1967; Schechter & Berger,
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1967). The catalytic domain is stabilized by two intradomain disulphide bridges and several conserved salt bridges. Multiple sequence alignment analysis indicated that 50 amino acids including three cysteine residues are strictly conserved. Human meprins are inhibited by hydroxamic acid derivatives such as Pro-Leu-Gly-hydroxamate which is also a therapeutic target for matrix metalloproteinases (MMPs) (Broder & Becker-Pauly, 2013). It has been shown by modeling that the hydroxamate-based inhibitor, Pro-Leu-Gly-hydroxamate, binds to the non-primed sites of human meprin-α and -β. The catalytic zinc is penta-coordinated with three histidine residues of human meprins (αHis90/βHis91, αHis94/ βHis95, and αHis100/βHis101), carbonyl oxygen of glycine, and ‘O’ atom of hydroxamate of the inhibitor in the complex. An important anchoring function in this interaction is played by the niche-like S3 subsite of the β-edge strand, which harbors the inhibitor’s proline ring (Bode et al., 1994; Grams et al., 1996). The hydroxamic acid derivative inhibitor modeled in the S1, S2, and S3 subsites of the active site cleft of human meprin-α and -β structure has been refined by explicit solvent MD simulations. Analysis of the MD refined complexes help in identifying the different interactions contributing to protein stability and structural flexibility at protein-inhibitor binding sites. The different residues at the P1, P2, and P3 positions of the inhibitor interacting with different amino acid residues in S1, S2, and S3 subsites of human meprin-α and -β helps in determining the substrate specificity of the two enzymes. 2. Materials and methods 2.1. Homology modeling and docking The structures of protease domain of human meprin-α and -β were first modeled using the X-ray crystal structure of astacin (1AST) at 1.8 Å resolution as the template. Multiple sequence alignment was performed with ClustalW (Thompson, Higgins, & Gibson, 1994) using a gap penalty of 10 and employing the GONNET matrix (Larkin et al., 2007) to know the conserved residues among the sequences. Sequence homology (above 30% identity) of the meprins with astacin was used to construct the homology models using Discovery Studio 3.5 (Accelrys Inc., San Diego, CA) based on MODELLER 9v10 (MartiRenom et al., 2000; Sali & Blundell, 1993). From the resultant 100 models generated, the best model was selected on the basis of lowest probability density function (PDF) score. The generated modeled protein was checked to correct any improper bond lengths, bond angles, conformations as well as add missing atoms and correct connectivity within the protein. The stereochemical quality of the models was validated by Ramachandran plot using PROCHECK (Laskowski, MacArthur, Moss, & Thornton, 1993; Morris, MacArthur, Hutchinson, & Thornton,
1992). Docking was done by superimposing the modeled meprins on the crystal structure of astacin complexed with Pro-Leu-Gly-hydroxamate (PDB ID:1QJJ). Solvent accessible surface area (SASA) can be used to predict the binding – induced conformational changes of the protein – inhibitor complexes. It was calculated by setting a probe radius of 1.40 Å and 240 grid points per atom using Discovery Studio 3.5. Two models of the protease domain of human meprin-α and -β in complex with Pro-Leu-Gly-hydroxamate inhibitor was used for MD simulations. 2.2. Molecular dynamic simulations All MD simulations were performed using Discovery Studio 3.5 with the CHARMm forcefield (Feller & Mackerell, 2000; Mackerell et al., 1998). Both models of human meprin-α and -β with hydroxamic acid derivative inhibitor was solvated with the TIP3P water molecules (Jorgensen, Chandrasekhar, Madura, Impey, & Klein, 1983) in an explicit spherical boundary condition (Brunger, Brooks, & Karplus, 1984) with a minimum distance of 7 Å from boundary to the protein atom. Each simulation was carried out by Standard Dynamics Cascade protocol of Discovery Studio 3.5, which includes two stages of minimization followed by heating, equilibrium, and production run. Positional constraints were imposed on the NE2 atom of three His residues at the active site with the zinc atom to avoid any drastic rearrangement of these residues. A distance cut-off of 10 Å from the inhibitor was used to select all atoms within that region encircling the inhibitor-bound active site of the protease domain on which dynamic simulation was run. All atoms outside the 10 Å region was fixed applying harmonic restraint. The solvated protease inhibitor models were first minimized using 500 steps of steepest descent followed by 1000 steps of conjugate gradient method to remove steric clashes and to better soak the water molecules into the macromolecules. This was done to remove any unfavourable contacts present in the models. The minimized structures were then subjected to heating from 50 to 300 K for 120 ps and equilibrated for 1 ns. Long-range electrostatic interactions were treated with a spherical cut-off method. Non-bond higher and lower cut-off distance were maintained at 12 Å and 10 Å respectively. Both complexes were further simulated for 20 ns at 300 K with a time step of 2 fs for production run. This was carried in the NVT ensemble. During the simulations, all covalent bonds involving hydrogen were constrained using the SHAKE algorithm (Ryckaert, Ciccotti, & Berendsen, 1977). The trajectories were saved every 50 ps interval for analysis. 2.3. Data evaluation The SASA was calculated to measure the differences of solvent accessibility of residues before and after inhibitor
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Comparative analysis of binding sites of human meprins binding. The root-mean square deviation (RMSD) of the protein backbone atoms from the initial coordinates was used as a measure for structural differences. The rootmean square fluctuation (RMSF) of the backbone atoms relative to the average structure over the last 5 ns simulations was calculated as an indicator of structural flexibility. RMSF gives a measure of the relative standard deviation for each residue in human meprins-α and -β. To find out the hydrogen bond stability of the protein-inhibitor complexes throughout the simulation period, we performed the heat map protocol (Dokla et al., 2012). Analysis of salt bridge was done using an oxygen–nitrogen distance cut-off of 5.0 Å between charged residue side chains. All the above analyses were performed with Accelrys Discovery Studio 3.5 (Accelrys, v 3.5.0.12158). 3. Results and discussion 3.1. Multiple sequence analysis Multiple sequence alignment of the protease domain of human meprins-α and -β (humep-α and humep-β) share a sequence identity of 30 and 37%, respectively, to astacin (Figure 1). Sequence alignment indicates that 50 residues including three cysteine are strictly conserved. Homology models built using the three-dimensional structure of astacin (PDB: 1AST) reveal structural similarity among the proteases. The models were sorted according to lowest PDF scores. Validation of the models show above 90% residues in allowed region of Ramachandran plot. Superposition of humep-α and -β models on the template astacin structure (1AST) show C–α RMSD values of 1.41 Å and 0.67 Å, respectively. 3.2. Protein – ligand binding We analyzed the intermolecular interactions of generated inhibitor docked structure of both human meprin complexes (Table 1). In case of humep-α complex, six
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intermolecular hydrogen bonds involving residues Cys63, Ser65, Glu66, and Tyr149 were detected. Apart from six H-bonds, electrostatic interactions involving two salt bridges between Glu66 of the protease and Proline residue of inhibitor were observed. In case of humep-β complex, five intermolecular hydrogen bonds involving residues Cys63, Ser65, Glu92, and Tyr150 were detected. Apart from these, one pi-cation interaction between Trp64 of the protease and Pro residue of inhibitor was also observed. In both human meprin complexes residues Cys63–Val67 forms the upper edge of the active site cleft, so that the carbonyl group of Cys63 presumably plays a role in hydrogen-bonding and polarizing the amino group of hydroxamic acid of bound inhibitor. The hydrogen bonds and electrostatic interactions add stability to the structures (Bella & Humphries, 2005; Xu, Tsai, & Nussinov, 1997). The total SASA is the surface of a biomolecule that is accessible to a solvent. The burial of hydrophobic amino acids in the protein is a driving force in protein folding. The extent to which an amino acid interacts with the solvent and the protein core is naturally proportional to the surface area exposed to these environments (Durham, Dorr, Woetzel, Staritzbichler, & Meiler, 2009). The SASA curve for complexed and non-complexed human meprins-α and -β is represented in Figure 2(a) and (b), and the residues that differ in solvent accessibility upon inhibitor binding are shown in Table 2. The SASA analysis of meprins before and after inhibitor binding reveals that there is decrease in solvent accessibility value for meprin residues interacting with hydroxamic acid derivative inhibitor. From the SASA plot, we observed that His90 that acts as a Zn-binding ligand in penta-coordinated structure of the metalloprotease domain is not involved in inhibitor binding in case of humep-α – inhibitor complex. The interacting residues found from SASA analysis are the ones involved in hydrogen bonding and other electrostatic interactions.
Figure 1. Multiple sequence alignment of protease domain of human meprin-α and -β with crystal structure of astacin (1AST). ‘*’ strands for identical residues, ‘:’ strands for conserved substitutions and ‘.’ strands for semi-conserved substitutions.
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Table 1.
Intermolecular interaction of docked complexes.
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Sl. No
Bond
Ligand residue
Receptor residue
Interaction constituents
Distance
(a) Humep-α – hydroxamic acid derivative complex 1 H bond1 LEU 2 H bond2 GLY 3 H bond3 HOA 4 H bond4 PRO 5 H bond5 HOA 6 H bond6 HOA 7 Salt bridge1 PRO 8 Salt bridge2 PRO
SER65 TYR149 TYR149 GLU66 CYS63 TYR149 GLU66 GLU66
SER65:N – LEU:O TYR149:OH – GLY:O TYR149:OH – HOA:O GLU66:OE1 – PRO:NHT1 CYS63:O – HOA:HN1 TYR149:OH – HOA:HO GLU66:OE1 – PRO:N GLU66:OE2 – PRO:N
2.3512 3.0414 2.364 2.3372 1.7426 2.4179 2.59634 4.31529
(b) Humep-β – hydroxamic acid derivative complex 1 H bond1 LEU 2 H bond2 HOA 3 H bond3 LEU 4 H bond4 HOA 5 H bond5 HOA 6 Pi-cation PRO
SER65 TYR150 SER65 CYS63 GLU92 TRP64
SER65:N – LEU:O TYR150:OH – HOA:O SER65:O – LEU:HN CYS63:O – HOA:HN1 GLU92:OE2 – HOA:HO TRP64 – PRO:N
2.7748 3.0393 1.984 1.817 2.274 5.7778
3.3. Global stability of the system Stability of the system was evaluated in terms of potential energy (PE) and total energy (TE) as a function of simulation time. Both the energy profiles are lower in case of humep-β complex and remained constant throughout 20 ns production run in both human meprin complexes (Figure 3(a) and (b)). 3.4. Protein structural features The meprin-hydroxamic acid stability in the 20 ns simulation was verified by monitoring the time evolution of structural parameters. The main chain RMSDs of modeled humep-α and humep-β – hydroxamic acid complexes (Figure 4) with respect to the initial structures as a function of time throughout 20 ns molecular dynamics simulation were analyzed to find deviations at the structural level. The production phase of dynamic simulation started at 1.12 ns after completing 120 ps of heating and 1 ns of equilibration. The results reveal that the RMSD plot of both humep complexes increased from the initial phase of the production run to 0.26 nm which remained more or less stable around 0.25–0.27 nm during the production run. For humep-β complex, the RMSD plot remained stable for the remaining period of the production analysis. In case of humep-α complex, the RMSD value increases and fluctuates with a larger amplitude of 0.29 nm at ~15.12 ns and remained stable at that value for the remaining 6.12 ns simulation period. Inhibitor binding is related to protein flexibility at the protein-ligand binding site. To understand the conformational flexibility of the protein-inhibitor complex at different binding site residues we evaluated the RMSF of the main chain as a function of residue number. Figure 5(a) and (b) show the specific residues of humep-α
and humep-β involved in Pro-Leu-Gly-hydroxamate binding. The results of the RMSF plot indicate that for humep-α – hydroxamic acid complex there were significant fluctuations around residues 60–69, 70–80, 86–95, 97–103, 121–127, 129–131, and 146–149, respectively. The inhibitor bound complex of humep-β exhibited fluctuations at residues 59–70, 73–81, 86–96, 98–105, 122–129, 131–133, 146–151, and 171–174, respectively. Residues α/βCys63, α/βTrp64, and αAsn125/βAsn126 comprise the S1 subsite of human meprins. S2 subsite consists of residues α/βSer65, αTyr99/βTrp100, and αAsp131/ βAsp132, and S3 subsite contains residues α/βTrp64 and αGlu66/βSer66. RMSF curves clearly indicate strong fluctuations in these non-primed subsite regions of the metalloproteases involved in inhibitor binding. A higher structural flexibility is observed at residue Gly68 having greater amplitude for humep-α complex (~0.25 nm) compared to humep-β complex (~0.15 nm). However, residues 63 and 64 comprising both S1 and S3 subsite region show same structural fluctuations for both complexes. Ser65 which is one of the S2 subsite residues, show greater fluctuations for meprin-α than meprin-β complex. In humep-α complex, greater fluctuations at residue Ser65 and Gly68 may be due to the presence of negatively charged amino acids, Glu66 and Asp69 adjacent to the respective amino acids, whereas the corresponding amino acid residues in humep-β complex are Ser66 and Asn69. In humep-α, Glu66 that forms the salt bridges with Proline residue of the inhibitor have greater fluctuation than corresponding Ser66 of humep-β complex. Greater structural flexibility at residues 146–149 is observed for humep-β complex (~0.18 nm) than humep-α complex (~0.12 nm). Additional fluctuation at residues 171–174 is reported for humep-β which is not found in humep-α complex
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Comparative analysis of binding sites of human meprins
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Figure 2. SASA plot of human meprin-α (a) and -β (b) before and after docking. Before docking is represented with blue line and after docking is represented with red line in the plot. Table 2.
Residues of human meprins that differ in solvent accessibility upon inhibitor binding.
Protein
Residues name
Humep-α-HA Humep-β-HA
Cys63, Trp64, Ser65, Glu66, Val67, Asn75, Glu91, His94, Tyr99, His100, His124, Tyr149 Cys63, Trp64, Ser65, Ser66, Val67, Arg71, His91, Glu92, His95, Trp100, His101, Tyr150
3.5. Hydrogen bonding In order to characterize the differences in conformational dynamics of the meprin-hydroxamic acid derivative complexes, we examined the hydrogen-bonding pattern in the 20 ns simulations. The stability of the
protein – ligand complex can be explored in terms of hydrogen bonding, hence we focused on the behavior of the hydrogen bonds present at the binding sites of the protein – inhibitor complexes. Majority of the hydrogen bonds involve main chain-side chain O⋯H–O
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Figure 3.
Stability of the system. (a). PE vs. Time (b). TE vs. Time.
Figure 4. Comparison of trajectory of the root-mean square deviation of human meprin-α and -β complexes with respect to its initial structure. Blue and Red line indicates humep-α and -β respectively.
and O⋯H–N hydrogen bonds. The hydrogen bond interactions of the last 5 ns average structure were obtained from MD simulations of both human meprin complexes (Table 3). It shows that some of the H-bonds present in the modeled structures of both meprin-inhibitor
complexes before simulation were lost and some new H-bonds were generated on completion of the 20 ns production run. Analysis of the stability of the H-bonds from last 5 ns average structure in both cases revealed that the H-bonds Glu66:OE1 – Pro:NHT1 and
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Comparative analysis of binding sites of human meprins
Figure 5.
Table 3. Sl. No
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Root-mean square fluctuations per residue of human meprin-α (a) and -β (b) hydroxamic acid derivative complexes. H-bond interaction of last 5 ns average structure. Bond
Ligand Residue
(a) humep-α – hydroxamic acid derivative 1 H bond1 LEU 2 H bond2 PRO 3 H bond3 LEU (b) humep-β – hydroxamic acid derivative 1 H bond1 LEU 2 H bond2 HOA 3 H bond3 HOA
Receptor Residue
Interaction Constituents
Distance
SER65 GLU66 SER65
SER65:HN – LEU:O GLU66:O – PRO:NHT1 SER65:O – LEU:HN
2.1534 1.7655 2.076
SER65 CYS63 GLU92
SER65:O – LEU:HN CYS63:O – HOA:HN1 GLU92:OE2 – HOA:HO
2.0334 2.3405 1.9556
Ser65:O – Leu:HN were present throughout the 20 ns simulation in case of humep-α complex. In case of humep-β complex, the two H-bonds Ser65:O – Leu:HN and Glu92:OE2 – HOA:HO persist throughout simulation. The Ser65 residue present at S2 subsite is involved in main chain – main chain hydrogen bonding for both the humep complexes. S3 subsite residue Glu66 of humep-α complex is involved in main chain – side chain hydrogen bonding with Proline of hydroxamic acid derivative. However, identical residue Ser66 present in the S3 subsite region of humep-β complex is not involved in hydrogen bonding with the inhibitor.
This indicates that P3 site of inhibitor has a preference for acidic residue at S3 subsite to form H-bond imparting stability to protein inhibitor complex. Hydrogen bond involving tyrosine residue present in the SXMHY domain (Met-turn) of the metalloproteases is observed before simulation but absent after simulation in both the cases. The last 5 ns average structure of both human meprin-inhibitor complexes show that the carbonyl oxygen of glycine and oxygen atom of hydroxamic acid of the inhibitor together with the three active site histidine residues of meprins maintain the penta-covalent geometry of the active site cleft (Figure 6).
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Figure 6. Hydrogen bonding network and Zn co-ordination of human meprin-α (a) and -β (b). Inhibitor shown in ball and stick and important residues present in active site shown in stick. Inhibitor coordinates the catalytic zinc ion (grey sphere) via carbonyl oxygen of glycine and ‘O’ atom of hydroxamic acid. Figure created with PyMol (www.pymol.org).
4. Conclusion In this article, we performed MD simulation to compare and analyze the substrate/inhibitor binding sites of human meprins-α and -β. An inhibitor Pro-Leu-Gly-hydroxamate was modeled into the active site cleft of both meprins following the mode of binding of the inhibitor in the active site of astacin. It is to be mentioned that studies on conformational flexibility as shown by RMSF calculation point out certain flexible regions in the
meprin structures, which may have an important role in imparting stability to the meprin-hydroxamic acid derivative complex. Analysis of simulation trajectories indicated that some H-bonds involving S1, S2, and S3 subsite residues of meprins Glu66:OE1 – Pro:NHT1, Ser65:O – Leu:HN, and Ser65:O – Leu:HN, Glu92:OE2 – HOA:HO, persist throughout the simulation period in humep-α and -β complexes, respectively. S2 subsite residue Ser65 present in both human meprin complexes
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Comparative analysis of binding sites of human meprins may have an important role in ligand binding as reflected from H-bonding analysis. Human meprin α and β have distinct specificities for preferred amino acids at or near the cleavage site due to subtle changes in amino acids in the corresponding S1, S2, and S3 subsites of the active site cleft which in turn to some extent explains the catalytic activity of the proteases. This knowledge is required for the structural alteration in the inhibition of some of these ligands in order to modify the biological properties of this protein. Further studies with other ligands binding to both primed and non-primed region of meprins could give more insight into the mode of action of meprin inhibitors. The information of receptor-ligand interaction is important and it guides the selections of candidature sites for further experimental data studies. List of Abbreviations HOA hydroxamic acid humep-α – HA human meprin-α – Pro-Leu-Gly-hydroxamate humep-β – HA human meprin-β – Pro-Leu-Gly-hydroxamate
Acknowledgement Our sincere thanks to Dr Asim Bera for his assistance in the preparation of Figure 6 using Pymol.
Funding We are thankful to the Department of Biotechnology (DBT), Government of India for their financial support.
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