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Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

Conformational Dynamics of Matrix Metalloproteinase-1·TripleHelical Peptide Complexes Tatyana G. Karabencheva-Christova,*,†,‡ Christo Z. Christov,*,†,‡ and Gregg B. Fields*,§,∥ †

Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne NE1 8ST, United Kingdom ‡ Department of Chemistry, Michigan Technological University, Houghton, Michigan 49931, United States § Department of Chemistry & Biochemistry, Florida Atlantic University, Jupiter, Florida 33458, United States ∥ Department of Chemistry, The Scripps Research Institute/Scripps Florida, Jupiter, Florida 33458, United States S Supporting Information *

ABSTRACT: Matrix metalloproteinase-1 (MMP-1) is a zinc-dependent protease that catalyzes hydrolysis of interstitial collagens. A previously reported X-ray crystallographic structure revealed specific interactions between a triple-helical peptide (THP) model of interstitial collagen and the hemopexin-like (HPX) and catalytic (CAT) domains of MMP-1. An NMR-based structure of MMP-1 in a complex with a different THP was also solved, where docking was used to model the MMP-1·THP interactions and develop a mechanism for the early stages of collagenolysis. To provide greater insight into and reveal specific details of the collagenolytic mechanism, molecular dynamics (MD) studies of the MMP-1·THP NMR-derived and X-ray crystallographic complexes were performed and compared. The “open/ extended” conformation of the NMR-derived MMP-1·THP complex was found to lead to a catalytically productive complex. The X-ray crystallographic MMP-1·THP complex was initially in a “closed/collapsed” conformation, and did not yield a productive complex. The NMR-derived structure of the MMP-1·THP complex possessed many more atomistic interactions between MMP1 and the THP compared with the X-ray crystallographic structure of the MMP-1·THP complex, and also had greater participation of MMP-1 in the local unwinding/destabilization of the THP. The atomistic interactions support the favorable energetics of the initial step of collagenolysis originating from the NMR-derived MMP-1·THP complex structure.

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“closed/collapsed” conformation, characterized by close orientation of the MMP-1 HPX and CAT domains.8 In addition, the MD study revealed that the linker region influenced interactions of the HPX and CAT domains with the THP.8 NMR spectroscopic and small-angle X-ray scattering (SAXS) analyses suggested an equilibrium between open and closed conformations of MMP-1 in solution.6,9,10 NMR spectroscopic analysis and docking calculations of MMP-1 in a complex with a type I collagen model THP indicated an initial “open/ extended” conformation of MMP-1 (Figure 1) and a resultant productive form of the MMP-1·THP complex.6,10,11 NMR spectroscopic analysis indicated that the MMP-1 HPX domain binds two strands of the collagen triple-helix simultaneously, allowing for a single strand to access the active site in the CAT domain.6

INTRODUCTION Matrix metalloproteinase-1 (MMP-1) is an enzyme that catalyzes the hydrolysis of collagen, the dominant protein component of the extracellular matrix in eukaryotes.1 MMP-1 is composed of a catalytic domain (CAT), a hemopexin-like (HPX) domain, and a linker region that connects the two domains (Figure 1). Collagenolysis is believed to be initiated by coordination of the carbonyl group of the scissile peptide bond to the active site Zn2+ located in the MMP-1 CAT domain.2−4 Experimental studies suggested sequential hydrolysis of single collagen strands, due to the relatively small active site volume of MMP-1 which is unable to host the intact collagen triplehelix.5,6 The X-ray crystallographic structure of MMP-1 complexed with a triple-helical peptide (THP) model of type II collagen described specific interactions of the individual strands of the THP with both the CAT and HPX domains.7 However, the lack of access of the THP leading strand to the MMP-1 active site made the X-ray crystallographic structure unproductive for performing catalysis. We performed molecular dynamics (MD) simulations of the MMP-1·THP X-ray crystallographic complex, and found that MMP-1 was in a © XXXX American Chemical Society

Special Issue: Ken A. Dill Festschrift Received: October 2, 2017 Revised: November 15, 2017 Published: November 21, 2017 A

DOI: 10.1021/acs.jpcb.7b09771 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. NMR-derived MMP-1·THP structure (Figure 4B from ref 6) drawn using UCSF (University of California San Francisco) Chimera.36 MMP-1 (displayed in silhouette round ribbon) consists of the CAT domain (blue), interdomain linker (spring green), and HPX domain (orange). The leading, middle, and trailing strands of the THP are shown in tube representation in cyan, green, and red, respectively. Zn2+ and Ca2+ bound to the enzyme are shown in spherical representation in green and magenta, respectively. Reproduced with permission from ref 6. Copyright 2012 American Chemical Society.

Figure 2. Sequences and numbering of THPs. Hyp = 4-hydroxy-L-proline.

numbering in the NMR-derived study included the 19 residue signal sequence,6 while the MMP-1 numbering in the X-ray crystallographic study did not.7 The present study follows the X-ray crystallographic study numbering for MMP-1. Atomistic MD simulations were performed using the Gromacs 4.5.5 package15−17 with the GROMOS96 43a1 force field.18 His149, His164, His199, His203, and His209 were protonated at their δ atom position (pdb atom name ND1) and His177 at the ε atom position (pdb atom name NE2). Protonation states for the rest of the titratable residues were assigned using the pdb 2gmx command in Gromacs. Parameters for Zn2+ and Ca2+ were obtained from the GROMOS96 43a1 force field,18 and the positions of both ions were kept restrained in the MD simulations using the harmonic potential. In order to remove steric clashes, the X-ray crystallographic and NMR-derived structures were subject to in vacuo energy minimization, using the steepest descent19 and conjugate gradient algorithms.20 The minimized protein structures were then placed in a cubic box (with a minimal distance of 10.0 Å from any edge of the box), and then solvated by using the single point charge (SPC) water model.21 The solvated structures were neutralized by adding 10 Cl− and 8 Cl− ions to the X-ray crystallographic and NMR-derived structures, respectively (the NMR-derived structure was missing a few residues near the MMP-1 N-terminus). Periodic boundary conditions were applied, and both systems were again energy minimized. MD of the restrained protein, allowing for movement of the solvent molecules, was performed in an NVT ensemble [constant number of particles (N), volume (V), and temperature (T)],22 at a constant temperature of 300 K, followed by NPT ensemble [constant number of particles (N), pressure (P), and temperature (T)] MD simulations for 50 ps.

The NMR-derived structure differs from the X-ray crystallographic structure in the initial mutual orientation of the CAT and HPX domains and their interactions with the THP. In the NMR-derived structure, the CAT domain is guided by the HPX domain, via the linker region, toward the scissile bond of the THP leading strand.6,10 Movement of the HPX and CAT domains, relative to each other, has been documented by both experimental and computational studies.12,13 In the present study, we examined and compared how interactions of the MMP-1 HPX and the CAT domains with the THP differ at the atomistic level in the NMR-derived and the X-ray crystallographic structures. Conformational flexibility is an inherent property of proteins, and MD simulations can provide vital information with regards to the flexible and dynamic nature of enzyme·ligand complexes.14 We performed atomistic MD simulations of the NMR-derived structure and the X-ray crystallographic structure of MMP-1·THP for 500 ns and characterized atomistic interactions in both structures.

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METHODS

The coordinates of the NMR-derived MMP-1·THP structure were obtained from Bertini et al.6 The coordinates of the X-ray crystallographic structure of MMP-1·THP (pdb 4AUO) were obtained from Manka et al.7 The NMR-derived structure utilized a type I collagen model THP, while the X-ray crystallographic structure utilized a type II collagen model THP (Figure 2).6,7 The THP numbering for the MMP-1·THP complexes in the present study was assigned on the basis of the sequence within the triple-helical regions of types I and II collagen, whereby the scissile bond is found at residues 775− 776 (Figure 2). The three strands within the THP are referred to as leading (L), middle (M), and trailing (T). The MMP-1 B


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Figure 3. RMSF analysis of MMP-1·THP. (A) RMSF analysis of the X-ray crystallographic (black) and the NMR-derived (red) structure of MMP-1· THP. (B) RMSF analysis of the linker region of the NMR-derived (red) and X-ray crystallographic (black) structures of MMP-1·THP.

X-ray crystallographic and NMR-derived structures equilibrated at approximately 50 ns, with an average RMSD value of 5.3 Å for the X-ray crystallographic structure and 6.7 Å for the NMRderived structure (Figure S1A). The overall RMSD trajectory (after 500 ns) of the NMR-derived structure showed a higher RMSD profile compared with the X-ray crystallographic structure (Figure S1A), indicating that the MD simulated NMR-derived structure has an increased structural deviation from the initial structure. As discussed earlier, NMR spectroscopic studies have demonstrated MMP-1 domain movement in solution.6,9,10 RMSD analysis of the individual domains of the X-ray crystallographic structure showed that the HPX domain had a greater structural deviation (RMSD = 2.7 Å) in comparison to the CAT domain (RMSD = 1.7 Å) (Figure S1B).27 The opposite was observed for the NMR-derived structure, as the CAT domain showed a greater structural flexibility in comparison to the HPX domain (RMSD = 4.2 and 2.3 Å, respectively) (Figure S1B). This increased structural deviation of the CAT domain in the NMR-derived structure might be an indication that it is positioning itself toward the THP, through the guidance of the HPX domain, during the MD simulation as hypothesized previously.6,10,11

Berendsen temperature coupling and Parrinello−Rahman pressure coupling were used to keep the system at 300 K, and 1 bar pressure was applied during position restrained dynamics. The productive MD simulation was carried out for 500 ns, using the NPT ensemble, with an integration time step of 0.002 ps. The Particle Mesh Ewald (PME) method23 was used for electrostatic interactions, and the Lennard-Jones potential was employed for the treatment of the van der Waals interactions, with the cut off distance set to 14.0 Å. The LINCS algorithm24 was utilized to keep all covalent bonds involving hydrogen atoms rigid. Analysis of the MD trajectories was carried out using the Gromacs 4.5.5 package and VMD (Visual Molecular Dynamics) software.25 The Bio3D package in R26 was used for principal component analysis (PCA) and dynamics domain cross correlation analysis (DDCCA).

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RESULTS AND DISCUSSION Conformational Dynamics of MMP-1·THP X-ray Crystallographic and NMR-Derived Structures. Rootmean-square deviation (RMSD) analysis of the Cα backbone atoms of the NMR-derived and the X-ray crystallographic MMP-1·THP structures was performed in order to assess the structural stability of each during the MD simulations. Both the C


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Figure 4. Principal component one (PC1) projections on the X-ray crystallographic and the NMR-derived structures of MMP-1·THP. (A) NMRderived structure. (B) X-ray crystallographic structure.

Å) compared with that of the X-ray crystallographic structure (33.5 Å) (Figure S2B). The linker region of MMP-1 consists of residues 243−258. In the NMR-derived structure, linker region residues 249−253 showed increased flexibility while residues 242−249, located in close vicinity to the CAT domain, showed reduced flexibility in comparison to the same residues in the X-ray crystallographic structure (Figure 3B). Residue Gly252 is involved in hinge bending,3 and its mutation results in enzyme activity reduction.28,29 In the MD simulation the backbone of Gly252 formed a hydrogen bond with the backbone of Arg281 that belongs to the “ball” region (residues Arg281, Phe282, and Phe297) of the HPX domain (Figure S4). PCA and Dynamics Cross Correlation Analysis (DCCA) of MMP-1·THP X-ray Crystallographic and NMR-Derived Structures. PCA analysis was performed to evaluate conformational motions for the NMR-derived and the X-ray crystallographic MMP-1·THP structures. The first three eigenvectors describe up to 58% and 49% of the overall variance in the NMR-derived and the X-ray crystallographic structures of the MMP-1·THP complexes, respectively. The first eigenvector showed that the THP moves toward blade I of the HPX domain in the NMR-derived structure (Figure 4A). Some linker region residues formed a coiled conformation, and moved toward the CAT domain. The HPX domain ball region moved in the direction to the CAT domain “socket” region (residues Gly214−Gln228). Importantly, the scissile bond of the THP L strand moved toward the active site pocket of the CAT domain. The HPX and the CAT domains in the NMR-derived structure also showed significant motion toward the THP.

The RMSD profile of the THP of the NMR-derived structure showed an increased structural deviation compared with the THP in the X-ray crystallographic structure (average RMSD = 8.8 and 7.0 Å, respectively) (Figure S1C). For MD simulations the THP was docked to the NMR-derived structure of MMP-1, and therefore, the increased structural deviation indicated an equilibration phase of the THP during dynamics. The structural stability of the docked THP was assessed by analysis of the intramolecular THP hydrogen bonding network, and hydrogen bonding interactions between the THP and the protein (Figure S2A). The number of hydrogen bonds between MMP-1 and the THP, and the intramolecular hydrogen bonds in the THP, equilibrate during the simulation and are indicative of the stability of the trajectory. The overall flexibility of both the X-ray crystallographic and the NMR-derived structures of MMP-1·THP was assessed using root-mean-square fluctuation (RMSF) analysis (Figure 3A). The MMP-1·THP NMR-derived structure exhibited increased flexibility in comparison to the MMP-1·THP X-ray crystallographic complex. More specifically, the flexibility of the CAT domain of the NMR-derived structure was increased compared to the flexibility of the CAT domain of the X-ray crystallographic structure. Blade I (residues 251−281; Figure S3) and blade IV (residues 402−412; Figure S3) of the HPX domain of the NMR-derived structure had increased flexibility compared to the same residues in the X-ray crystallographic structure. HPX domain blade I and blade II (residues 291−341; Figure S3) have been shown to interact with the THP.6,10,11 The distance between the centers of mass for the CAT and HPX domains was greater in the NMR-derived structure (37.9 D


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The Journal of Physical Chemistry B The THP strands in the X-ray crystallographic structure exhibited very limited motion toward the HPX domain; instead, motions were mainly directed toward the center of the THP (Figure 4B). The X-ray crystallographic structure linker region residues displayed limited motion toward the CAT domain, and there was no coil conformation formation. The overall amplitude of motions of the CAT and HPX domains was low in the X-ray crystallographic structure in contrast to the NMR-derived one. The radius of gyration of the THP in the NMR-derived structure showed an average value of 28.0 Å in comparison to 23.6 Å for the X-ray crystallographic structure (Figure 5). An

Figure 5. THP radius of gyration within MMP-1·THP of the X-ray crystallographic (black) and NMR-derived (red) structure. Figure 6. Cluster analysis of MMP-1·THP complex. The NMRderived structure of MMP-1 and the THP are shown in light blue and green, respectively. The linker region of the NMR-derived structure is shown in blue. The X-ray crystallographic structure of MMP-1 and the THP are shown in light pink and gold, respectively. The linker region of the X-ray crystallographic structure is shown in pink. (A) Superimposed MMP-1·THP complexes of the NMR-derived and Xray crystallographic structures. (B) Superimposed enzymes alone. (C) Superimposed THPs alone.

average structure comparison (based upon analysis of the most populated cluster) of the NMR-derived and the X-ray crystallographic structure showed significant conformational fluctuations in the THP and the linker region of the NMRderived structure (Figure 6). In particular, a local unwinding of the THP and a formation of a coil in the linker region was observed. The NMR-derived structure clearly showed an HPX domain perturbation effect exerted on the THP strands. These results indicated that in the NMR-derived structure the THP experienced less compression compared to the X-ray crystallographic structure.27 The angle measured for the X-ray crystallographic and NMR-derived structures indicated significant differences in the orientation of the THP. The THP L strand of the X-ray crystallographic structure showed a significant bending of 86.1°, while in the NMR-derived structure the bending angle was 122.9° (Figure S5). The different orientation of the CAT and HPX domain in the NMR-derived structure, compared with the X-ray crystallographic structure, influenced the orientation of THP. DCCA of the NMR-derived structure showed an overall increase in the anticorrelated motions in comparison with the X-ray crystallographic structure (Figure 7). More anticorrelated motions toward the HPX domain were detected in the CAT domain of the NMR-derived structure. For example, anticorrelated motions between the α-helix C and loops from the CAT domain and β-sheets 16, 17, 18, and 19 from the HPX domain were observed in the NMR-derived structure. The increased anticorrelated motions between the CAT and HPX domains suggest a more open conformation in the NMRderived structure compared with the X-ray crystallographic one. A previously observed specific correlated motion in the L338A/ H339A MMP-1 double mutant, which was based on the X-ray

crystallographic structure,8 does not appear in the NMRderived structure. The THP in the NMR-derived structure showed overall reduction in both positive and negative correlated motions, toward each other, compared to the Xray crystallographic structure. Atomistic THP/CAT Domain Interactions in MMP-1· THP X-ray Crystallographic and NMR-Derived Structures. The distance between the oxygen of the THP L strand scissile bond and the catalytic Zn2+ in the NMR-derived structure showed an average value of 4.7 Å in comparison to 6.7 Å for the X-ray crystallographic structure (Figure 8). In the NMR-derived structure the backbone of Gln167 in the CAT domain is hydrogen bonded to the backbone of Hyp771(L) and Gly772(L) of the THP. In the X-ray crystallographic structure, the backbone of Gln167 is involved in hydrogen bonds with the side chain of Hyp771(L) and Gly769(T) of the THP. In the NMR-derived structure the backbone of Ala165 was hydrogen bonded to the backbone of Gln774(L) up to 350 ns, and then the side chain of His209 stabilized the Gln774 residue after 350 ns (Figure 9). In the X-ray crystallographic structure His209 formed hydrogen bonds with the side chain of Gln774(L) while A165 had no interactions with the THP. The carbonyl group of Ala165 has been proposed to be involved in E


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Figure 7. Dynamics cross correlation analysis (DCCA) of (top) NMR-derived and (bottom) X-ray crystallographic structure of MMP-1·THP complex. The scale of correlated motion ranges from +1 to −1 and represents positive (red) and negative (blue) motions of Cα atoms.

crystallographic structure Asn152 made limited interactions with the side chain of Hyp771(M). The side chain of Ser153 in the X-ray crystallographic structure formed hydrogen bonds with the side chain of Hyp771(T). In the NMR-derived structure the side chain of Ser153 formed hydrogen bonds with the backbone of Gly769(T). The side chain of Hyp771 formed hydrogen bonds with the backbone of Pro154 of the CAT domain in the NMR-derived structure, while Pro154 of the X-

MMP-1 catalyzed collagen hydrolysis by stabilizing the positive charge at the nitrogen of the scissile bond.30 The residues in the vicinity of the scissile bond of the THP L strand are stabilized to a greater extent in the NMR-based structure compared with the X-ray crystallographic structure. In the NMR-derived structure the backbone of Hyp768(T) was stabilized by hydrogen bonding with the backbone of Asn152 of the CAT domain. However, in the X-ray F


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Figure 10. Interactions of Arg780(L) with the socket region of the Xray crystallographic structure.

Figure 8. Scissile bond distance to the catalytic Zn2+ in the NMRderived (black) and X-ray crystallographic (red) structures.

interactions with the hydrophobic component of the Arg780 side chain, with an average distance of 4.5 Å in the NMRderived structure. No interactions of Val300 were detected in the X-ray crystallographic structure. HPX domain blade I and blade II residues in the NMR-derived structure exhibited extensive interactions with the THP, which is in a good agreement with the NMR-derived structure itself.11 The Asn336 side chain stabilized the backbone of Gly784(L) after 400 ns (Figure 11). In contrast, in the X-ray crystallographic

Figure 9. THP L strand interactions with CAT domain residues for the NMR-derived MMP-1·THP structure.

ray crystallographic structure made no interactions with the THP. The backbone of Asp156 stabilized the side chain of Gln774(T) via hydrogen bonds in the NMR-derived structure, while in the X-ray crystallographic structure the side chain of Asp156 formed hydrogen bonds with the side chain of Gln774(T). In general, CAT domain residues of the NMRderived structure exhibited more interactions with the THP compared with the X-ray crystallographic structure. THP residues had interactions with the socket of the CAT domain in both the NMR-derived and the X-ray crystallographic structures. In the X-ray crystallographic structure, the THP Arg780 side chain and backbone interacted with the socket region Tyr218 and Pro219 residues, respectively, of the CAT domain (Figure 10). Similar results were obtained for the NMR-derived structure, where Arg780(L) formed hydrogen bonds with the side chain of Tyr218. Residues of the THP L and M strands also formed hydrogen bonds with the backbone atoms of the socket residues. Atomistic THP/HPX Domain Interactions in MMP-1· THP X-ray Crystallographic and NMR-Derived Structures. Most interactions of the HPX domain residues with the THP appear after 350 ns, and the RMSD of the NMR-derived structure shows conformational changes around the same time (Figure S1). This might be due to the fact that the THP was docked to the NMR structure6 and requires a longer time for equilibration. The side chain of Val300 assumed hydrophobic

Figure 11. Interactions of the side chain of Asn336 from the HPX domain blade II with the backbone of Gly784(L) based on the NMRderived structure.

structure the side chain of Asn336 pointed away from THP and Asn336 made no interactions with THP. The side chain of Leu338 made no interactions with any THP residues in the Xray crystallographic structure, while in the NMR-derived structure the side chain of Leu338 interacted (at 3.8 Å) with the side chain of Val783(L). In similar fashion, the side chain of His339 also formed hydrogen bonds near the end of the simulation (of the NMR-derived structure) with the side chain of Hyp786(L). In the NMR-derived structure the Gln335 side chain hydrogen bonded with the backbone of Leu785(L) until 350 ns, and then with the Val782(T) (Figure 12). In contrast, the side chain of Gln335 in the X-ray crystallographic structure made no interaction with the THP, and instead formed hydrogen bonds with the backbone of Phe301 of the HPX domain. The interactions in the NMR-derived structure pulled G


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with Pro788, Hyp789, and Gly790 from the THP T strand (Figure 13). In the X-ray crystallographic structure, Arg272

Figure 12. Interactions of the side chain of Gln335 from the HPX domain blade II with the leading and trailing strand of the THP based on the NMR-derived structure.

Figure 13. Interactions of the THP L strand with the HPX domain residues for the NMR-derived MMP-1·THP for 500 ns trajectory. O = Hyp.

the leading strand toward the loop region between β13−β14 of blade II and seemed to be causing local changes in the THP. These results are consistent with the NMR studies which indicated an extensive line broadening in the region Gly334− Val337.11 NMR studies showed that the interaction of the HPX domain with Val782, Val783, Gly784, and Leu785 of the THP L strand allowed for the correct positioning of the CAT domain for hydrolysis of the peptide bond.6 In the X-ray crystallographic structure, the backbone of Leu295 makes consistent hydrogen bonds with the side chain of Arg780(L). The side chain of Leu295 in the X-ray crystallographic structure is also involved in a hydrophobic interaction (average distance 4.1 Å) with the side chain of Ile213, which is located near the socket region of the CAT domain. Mutation of Leu295 reduced the enzymatic activity of MMP-1, 7 and thus, the specific interactions of Leu295 identified here may participate in MMP-1 catalyzed collagenolysis. The THP M strand showed a few interactions with the HPX domain in the NMR-derived structure. For example, the side chain of Glu294 participated in electrostatic interaction with the side chain of Arg780(M). However, in the X-ray crystallographic structure, the side chain of Glu294 interacted with Arg780(L). In the NMR-derived structure the side chain of Asn296 stabilized the backbone of Arg780(M) and the side chain of Arg780(L) via hydrogen bonds (Figure S6). The side chain of Asn296 also formed hydrogen bonds with the backbone of Gly781(M). In contrast, in the X-ray crystallographic structure, the side chain of Asn296 only hydrogen bonded with the backbone of Gly781(L). Glu294 and Asn296 in the X-ray crystallographic structure were involved in interactions with the THP L strand, while the same residues in the NMR-derived structure had interactions with both the L and M strands of the THP. The backbone of Phe301 formed hydrogen bonds with the backbone of Val783(M) in the NMRderived structure while no interactions were made by Phe301 with any strands of THP in the X-ray crystallographic structure. The side chain of Glu314 formed hydrogen bonds with the side chain of Hyp789(M) in the NMR-derived structure, while in the X-ray crystallographic structure Glu314 made no interaction with any of the strands of the THP but instead was involved in interactions with residues of the HPX domain. In the NMR-derived structure, Arg272 formed hydrogen bonds

formed hydrogen bonds with residues of the THP M strand. Mutation of Arg272 in MMP-1 caused a significant reduction in collagenolytic activity.7,31 In the NMR-derived structure, Ile271 of the HPX domain was involved in hydrophobic interactions with Phe301, Phe323, and the side chain of Leu785(M) (Figure 14). Mutation of Ile271 in MMP-1, in combination with mutation of Arg272, caused a reduction in collagenolytic activity beyond that of Arg272 mutation alone.31

Figure 14. Interactions of Ile271 in the NMR-derived structure of MMP-1·THP.

There were 15 residues from the MMP-1 HPX domain previously chosen for mutation based on NMR analysis.11 These residues were located in either blade I or blade II of the HPX domain and were proposed to be involved in binding to the THP. Therefore, it would be important to study these residues in the NMR-derived structure. The side chain of Asp280 interacted with the side chain of Arg281 with an average distance of 5.4 and 5.9 Å in the NMR-derived and Xray crystallographic structures, respectively (Figure S7). In the NMR-derived structure the side chain of Asp280 was also stabilized by a hydrogen bond to the backbone of Arg281. Interestingly, after 350 ns the backbone of Arg281 formed hydrogen bonds with Gly252 of the linker region and H


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Figure 15. Superposition of the closed/collapsed, X-ray crystallography-derived (beige) and open/extended, NMR-derived (light blue) conformations of MMP-1.

proposed to provide access of MMP-1 to the collagen triplehelix.32 Once accessed, two mechanisms for the initial stages of MMP-1 catalyzed hydrolysis of the triple-helix have been proposed. The first, on the basis of an NMR-derived structure of MMP-1 interaction with a THP, indicated an open conformation of MMP-1 initially (Figure 15), whereby the interactions between the CAT and HPX domains was minimal.6 The second, on the basis of X-ray crystallographic analysis of an MMP-1·THP complex, started with a closed/ collapsed conformation of MMP-1 initially (Figure 15).7 Presently, the THP L strand comes closer to the active site pocket during MD simulations of both the NMR-derived and the X-ray crystallographic structures. THP unwinding/destabilization was observed to a greater degree in the NMR-derived structure compared with the X-ray crystallographic structure, and unwinding/destabilization near the scissile bond of the THP was only observed in the NMR-derived structure. In the NMR-derived structure, interactions of Val782, Val783, Gly784, and Leu785 of the THP L and T strands were responsible for the local unwinding/destabilization of the THP, allowing for the access of the single chain of collagen to the enzyme active site. During the MD simulation of the X-ray crystallographic structure, both the HPX and CAT domains interact with the THP; however, there was no indication of collagen destabilization/perturbation near the scissle bond, in contrast with experimental studies.5,6,33 Residues of the THP L and T strands from the NMR-derived structure showed a significant number of interactions with the CAT domain, while the THP M strand had very few interactions with the CAT domain. Interactions of the THP L and T strands with the CAT domain strained these two strands and allowed for opening near the scissile bond. The scissile bond in the NMR-derived structure was properly positioned in the catalytic site of the CAT domain in contrast to its position in the X-ray crystallographic structure, supporting the proposal that the NMR-derived structure represents a catalytically productive form of the complex. MD simulations indicated a productive, feasible mechanism for collagenolysis starting from the NMR-derived structure, but

compressed the linker region (Figure S7). There were no hydrogen bonds between Arg281 and Gly252 in the X-ray crystallographic structure. The side chain of Arg281 formed hydrogen bonds with the backbone of Ser224 in both the NMR-derived and X-ray crystallographic structures. The side chain of Phe282 was stabilized by a hydrophobic cluster created by Phe297, Leu295, and Ile213 in the NMR-derived structure. Similarly, in the NMR-derived structure, the side chain of Phe297 was stabilized by residues Phe282 and Ile213. The aliphatic side chain of Arg281 also stabilized the aromatic ring of Phe297. The side chain of Ser299 formed hydrogen bonds with the side chain of Asn307. The side chain of Val300 stabilized the aliphatic chain of Arg780(L), with an average distance of 4.9 Å. Atomistic THP Interactions with Multiple Domains in MMP-1·THP X-ray Crystallographic and NMR-Derived Structures. In the X-ray crystallographic structure, the THP created an extensive interaction surface spanning nearly 60 Å in length.7 Complex formation with MMP-1 resulted in burying 480 Å2 of solvent accessible surface of the L strand, 440 Å2 of the M strand, and 370 Å2 of the T strand.7 The backbone of Arg780(L) was previously found to be hydrogen bonded to the backbone of Pro219 and Tyr221 of the CAT domain socket region.7 In the NMR-derived structure the Arg780(L) side chain interacted with the side chains of Tyr218 and Ser220 of the CAT domain and Leu296 of the HPX domain. Overall, we observed that Arg780(L) makes more interactions with the CAT domain in the NMR-derived structure and there was no bending of the THP in this structure. The increased flexibility of and key distances between the L, M, and T chains of THP (Figure S8) and the increased fluctuations in the bending angles of those chains after 350 ns (Figure S5), while not representative of an advanced separation process of the strands, would be part of an initial local unwinding event leading to this process.

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CONCLUSIONS Several recent studies have provided insight into the processing of fibrillar collagen. Strain-induced buckling of fibrils has been I



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not starting from the X-ray crystallographic structure. It was suggested in the X-ray crystallographic study that a different form of the MMP-1·THP complex may exist, whereby a rotation of the triple-helix places Leu785(L) (instead of the observed Leu785(M)) in the MMP-1 S10′ subsite and Leu776(T) nearby the MMP-1 S1′ subsite.7 However, residues 776−781 from the T strand must then be “stretched” and residues 773−775 reoriented for the T strand to insert into the MMP-1 active site, which requires breaking 4 interchain hydrogen bonds. The process of breaking 4 hydrogen bonds might be unfavorable energetically. In contrast, starting from the NMR-based MMP-1·THP complex and proceeding to the initial step of collagenolysis was found to be energetically favorable6 even considering that the HADDOCK energy function used in the calculations was highly simplified.34 The present study has revealed substantial conformational dynamics in both MMP-1 and the THP. Experimental and computational studies have shown that the HPX and the CAT domains of MMPs experience conformational changes with respect to each other.12,13 In concert with THP dynamics, a relationship between substrate dynamics and reaction rates has been reported in other proteolytic systems.35 Regardless of the THP conformational dynamics, MMP-1 was still required to “act” on the THP to allow for proper orientation of individual strands prior to catalysis, consistent with conclusions from prior studies.6,33,34

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b09771. RMSD of Cα atoms of the MMP-1·THP complex; hydrogen bonding profile of the MMP-1·THP NMRderived structure; the average distance between the centers of mass of the CAT and HPX domains; location of blades I, II, and IV in the MMP-1 HPX structure; snapshot of the 500 ns time frame; THP bending angle; interactions of HPX domain residues with the THP M strand; Asp280 and Arg281 interactions; and interchain distance within the THP (PDF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (T.G.K.-C.). *E-mail: [email protected] (C.Z.C.). *E-mail: gfi[email protected] (G.B.F.). ORCID

Gregg B. Fields: 0000-0003-3573-1527 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge support from the Marie Curie International Career Development Fellowships (T.G.K.C., C.Z.C.), National Service for Computational Chemistry Software (NSCCS) (T.G.K.C., C.Z.C.), High-End Computing (HEC)-BioSim (T.G.K.C., C.Z.C.), and National Institutes of Health CA098799 (G.B.F.), and assistance from Dr. Warispreet Singh on the MD simulations. Supercomputer resources and services used in this work were provided by the Northumbria University HPC-Cluster “Pasteur”. J


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