Insight towards the conserved water mediated recognition of catalytic and structural Zn+2 ions in human Matrix Metalloproteinase-8 enzyme: A study by MD-simulation methods

Human matrix metalloproteinase-8 (hMMP-8) plays a important role in the progression of colorectal cancer, metastasis, multiple sclerosis and rheumetoid arthritis. Extensive MD-simulation of the PDB and solvated structures of hMMP-8 has revealed the presence of few conserved water molecules around the catalytic and structural zinc (ZnC and ZnS) ions. The coordination of two conserved water molecules (W and WS) to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvement of that metal ion in the catalytic process. Beside this the coupling of ZnC and ZnS metal ions (ZnC – WH (W1)…..W2 ….H162 - ZnS) through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational on the recognition of two zinc ions which were separated by ~13 Å in their X-ray structures. This unique recognition of both the Zn+2 ions in the enzyme through conserved water molecules may be implemented/ exploited for the design of antiproteolytic agent using water mimic drug design protocol.


Background:
The matrix metalloproteinases (hMMPs) of human are zinc containing endopeptidases and they play some important roles in ovulation, embryogenic growth, tumor growth, metastasis, multiple sclerosis, rheumetoid arthritis etc [1]. The human neutrophil collagenase (HNC) or hMMP-8 is mainly produced by neutrophils and involves in colorectal cancer [2]. The biological role of hMMP-8 and its actual function / mechanism is incompletely understood. So, hMMP-8 is one of the attractive target for design the inhibitors of extracellular matrix degradation in colorectal cancer and antiproteolytic drug for inflammatory diseases [1,3]. The hMMP-8 is structurally homologous to all other MMPs, consisting of defined functional domains. The crystallographic structures (1BZS, 1JAP, 1ZP5, 1ZVX, 1ZSO) [4-8] of hMMP-8 Table 1 (see supplementary material) have explored the presence of two (catalytic and structural) zinc ions in catalytic domains of enzyme. Catalytic zinc ion (ZnC) is bound to three histidine reidues (H197, H 201, H207) and different inhibitors (BSI250, HOA4, 2NI128, FIN994, EIN994 respectively), whereas the structural zinc (ZnS) is coordinated by three histidine residues (H147, H162, H175) and one aspartic acid (D149) which are given in Table 2 (see supplementary material). The catalytic domain also has two calcium ions which are thought to involve in the structural function of enzyme. Furthermore, regarding the mechanism of activity of different hMMPs, the catalytic role of ZnC is well known [9]; however, the participation of other zinc ion (ZnS) in the catalytic process has also been indicated in MMP-13 [10].
In this work we perform long time MD simulations in order to study the dynamics of water molecules and their interaction with catalytic and structural zinc (ZnC and ZnS) centers. Beside these, the role of ZnS (structural zinc ion) in the catalytic mechanism / process of hMMP-8 can also be investigated through computational dynamic of solvated enzyme. In addition the simulation could also shade some light on the water mediated recognition between the two zinc centers and their conjugal involvement in the catalytic process. All the results may provide further complementary information on the inhibitor binding chemistry and also enrich the biochemical insight of hMMP-8 enzyme which may be useful for the development of inhibitor topology of hMMP-8.

Solvation
All the hMMP-8 structures were solvated using the program CHASA (Conditional Hydrophobic Accessible Surface Area) [16] without implying Periodic Boundary Condition (PBC). The explicit hydration have been employed on each structure where approx ~ 800-1000 water molecules have been used to immerse molecules by TIP3 water model.

Energy minimization of the structures
All the five modified PDB and solvated structures were energy minimized by GROMOS 96 force field [17] (500 steps of steepest decent followed by 1000 cycles of conjugate gradient) in the Swiss PDB Viewer program with 10Ǻ cut-off distance [18] for nonbonded interaction, using a distance dependent dielectric constant. At the initial stage of energy minimization, the protein was kept fixed and the water molecules were only allowed to move freely. Optimization released the local constraints from the structures, the energy minimization was again followed (1000 cycles) by allowing the protein and water molecules to move freely.

Molecular dynamics structures
For investigating the role of water molecules, solvation of native PDB structures was done by creating a water box of dimension 10 × 10 × 10 Ǻ 3 , using solvate 1.2 plugin within the Visual Molecular Dynamics v. 1.8.6. program [19] and MD simulations were followed. Total ~ 2196 water molecules (TIP3P water model) were used to immerse each molecule. The molecular dynamics of each structure was performed by converting the solvated and PDB structures to Protein Structure File (PSF) using the tool Automatic PSF Generation Plugin-1.0 v by applying CHARMM force field [20, 21] within the Visual Molecular Dynamics v. 1.8.6. All the ten (five PDB and their solvated) structures were simulated. All the structures were initially energy minimized (100 cycles to eliminate initial contacts which would destabilize the system) using CHARMM force field. After energy minimization all the structures were simulated using Auto Interactive Molecular Dynamics (IMD) v.1.8.6 and Nanoscale Molecular Dynamics v.2.6. [22]. During simulation, the whole system was allowed to move freely [23]. The molecular simulations were followed upto 5 ns for each structure (where time step was 2fs) at 300K temperature by means of Langevin dynamics [24]. The whole system was converged within 500ps and the simulation was adequately converged within 2ns (Figure 1). During the dynamics, several snapshots were recorded (every 2ps) to investigate the detail coordination or interaction of water molecules with the Zinc (ZnC / ZnS) ions.

Identification of conserved water molecules
To identify the conserved water molecules in hMMP-8, the five different X-ray crystal structures of the protein (1BZS, 1JAP, 1ZP5, 1ZVX, 1ZSO) were compared using 1BZS as template. All the other structures were superimposed one by one on the template structure by a standard least-square fitting algorithm using Swiss PDB Viewer program. The fit was initially optimized on the backbone atoms and the RMSD values were ~0.5-0.6Ǻ. After superimposing the concerned structure on the template, the conserved water molecular sites were compared and located between the two respective structures. Water molecules that were found to be within 1.5Ǻ [25,26] in between the two structures (template and concerned reference structures) were taken as conserved.

Binding free energy and electrostatic free energy of Zn ions
Binding free energy and electrostatic free energy of both the Zn ions in the PDB and MD simulated hMMP-8 structures were calculated by FOLDX program [27]. The temperature, ionic strength, pH and VDW (parameters of FOLDX) were assigned as 300K, 0.05 (M), 7.0 and 2.0 Ǻ.

Results: X-ray Structural information
In this work the catalytic and structural zinc ions are designated by ZnC and ZnS. Throughout the work the water molecules which are directly coordinated to ZnC are abbreviated as WH and WI. The WH (Trans to ZnC bound H207) is thought to involve in the hydrolysis of substrate [28]. The WI (Trans to ZnC bound H197) is in close proximity to inhibitor binding site. In the five PDB structures coordination of residues (H197, H 201, H207) to ZnC is same. In these structures ZnC is also bonded to a competitive inhibitor…. (BSI250, HOA4, 2NII8, FIN995, EIN994 respectively). In the structures 1JAP, 1ZVX, 1ZSO the inhibitor molecule has two donor centers, whereas in 1BZS and 1ZP5 the inhibitors have only one donor center. However, the structural zinc ion (ZnS) is coordinated by three histidines (H147, H162, H175) and one aspartic acid (D149).

Analysis of energy minimized structures
During energy minimization of all the PDB structures, the coordination number of ZnC changes due to the bonding of incoming water molecules to this metal ion. In the energy minimized native 1BZS, 1JAP, 1ZVX crystal structures, two incoming water molecules (WH and WI) form bond with ZnC, and WH also forms H-bond to E198 (2.5 to 3.5 Ǻ). Whereas in the PDB structures of 1ZP5 and 1ZSO only one incoming water molecule (WI) is bonded to ZnC. However, no such water molecular interaction is observed at ZnS. All the distances are included in Table 3 (see supplementary material). Again, after energy minimization of the two (1BZS, 1JAP) solvated PDB structures two incoming water molecules are observed at the same hydrophilic WH and WI sites which are included in Table  3. In the rest three (1ZP5, 1ZVX, 1ZSO) solvated structures, only one water molecule appears at WI site and coordinated to ZnC. However no water molecular coordination is observed at the structural zinc ion (ZnS).

Analysis of MD simulated structures
MD simulation of the different X-ray and solvated structures has also revealed the presence of water molecules at the WI and WH hydrophilic centres. Within 500ps, both the water molecules (present at the two hydrophilic centres) and one glutamic acid residue (E198) are appeared to coordinate with ZnC. In every structure the WH water molecule forms H-bond to E198 (OE1) (~ 2.1 to 2.6 Ǻ). During 5ns simulation, the trigonal geometry around ZnC has changed to distorted octahedral and the coordination number changed from 3 to 6 Table 4 (see supplementary material) (Figure 2). In the simulated PDB structures, the average ZnC …..W1, W1…..W2 and W2…..H162 distances are 2.07 -2.17, 2.14 -2.80 and 2.23 -2.87Ǻ respectively. In all the simulated X-ray and solvated structures of hMMP-8 (1BZS, 1JAP, 1ZP5, 1ZVX, 1ZSO) the ZnS atom is coordinated by two positionally conserved water molecule (W and WS) and three histidine and one aspartic acid residue. Among the two water molecule one (WS) also bonded to S151. Within 500ps, these two water molecules appear to coordinate with ZnS and S151 also bonded to WS. In the simulated X-rays structures the average ZnS …..W and ZnS…..  Table 6.

Discussion:
The hydrophilic center around both the zinc ions (ZnC and ZnS) in hMMP-8 structures seems to be interesting. The coordination of water molecules present at WH hydrophilic site which forms H-bond to E198 around ZnC ion in three Emin X-ray structures (1BZS, 1JAP, 1ZVX) and all MD simulated structures are observed to be conserved. Furthermore, the coordination of WI water molecule in two Emin X-ray structures (1ZP5, 1ZSO) and all MD simulated structures is also conserved. The presence of WH and WI hydrophilic centers has been revealed during the simulation of hMMP-8 PDB and solvated structures. These two conserved water molecules may provide some stability to ZnC ion. Nevertheless, in all the MD simulated X-ray and solvated structures, the ZnS bound two water molecules occupying the same hydrophilic centers (trans to H162, H175), indicate the possibilities of variable hydration susceptibility of catalytic and structural zinc ions (ZnC and ZnS) in the hMMP-8 dynamical structures Table 4. The shift or fluctuation of binding and electrostatic free energy of both the zinc ions in the X-ray and simulated structures may imply some possibility of ZnS centre to act as a catalytic partner.
The variation of coordination geometry from tetrahedral to distorted octahedral through the interaction of two water molecule (W and WS) directly to ZnS in all the PDB and solvated MD-simulated structures and the H-bonding of WS with S151(OG) Table 7 (see supplementary material) indicated the possible involvement of metal ion and S151 to the catalytic process. Furthermore, the recognition of the ZnC and ZnS (which are ~ 13Å apart) through two conserved water molecules (WH / W1 and W2) and the observation of similar type of water mediated conjugation of metal ions in different synthetic Zn-complexes [29,30] may also support the role of two Zn centres in the function and mechanism of enzyme ( Figure 2 & 3). Based on the dynamical results of this work the participation of ZnS in the collagenolysis mechanism of hMMP-8 may be proposed (Figure 3). The ZnS directly binds to substrate and polarize it and subsequently activate the water molecule which may then act as a nucleophile. The ZnS bound WS (water molecule) and S151(OG) may also thought to activate the metal ion in the intermediate transition steeps of the photolytic mechanism.

Conclusion:
The MD-simulation of the PDB and solvated structures of hMMP-8 have revealed the presence of few conserved water molecules (W and WS) around the catalytic and structural zinc (ZnC and ZnS) ions, which are mostly inaccessible in X-rays structures. The coordination of these two conserved water molecules to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvement of that metal ion in the catalytic process. The differential coordination dynamics of the ligands (water molecules) around both the zinc ions seem to be an interesting feature of hMMP-8. Beside this the coupling of ZnC and ZnS metal ions through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational clues on the recognition of two zinc ions which were separated by ~13 Å in their X-ray structures. Possibly, the biochemical signal between the two Zn +2 ions may be transmitted through W1 and W2 water centers in enzyme.
The unique recognition and stereospecific interaction of both the Zn +2 ions through conserved water molecules of both the Zn +2 ions in the enzyme (ZnC -WH (W1)…..W2 ….H162 -ZnS) seem to be important for activity and ligand recognition. Possibly, the geometrical and electronic consequences of conserved water molecular interaction W and WS are very implicative for design the topology of hMMP-8 inhibitor which may be implemented / exploited for the design of antiproteolytic agent using water mimic drug design protocol. Possibly, heterocyclic ligand with flexible structure containing two aliphatic short chains with suitable spacer length may be suggested to design selective inhibitor for hMMP-8.