Importance of the linker region in matrix metalloproteinase-1 domain interactions †

Collagenolysis is catalyzed by enzymes from the matrix metalloproteinase (MMP) family, where one of the most studied is MMP-1. The X-ray crystallographic structure of MMP-1 complexed with a collagen-model triple-helical peptide (THP) provided important atomistic information, but few details on the e ﬀ ects of the conformational ﬂ exibility on catalysis. In addition, the role of the linker region between the catalytic (CAT) and hemopexin-like (HPX) domains was not de ﬁ ned. In order to reveal the dynamics and correlations of MMP-1 comprehensive atomistic molecular dynamics simulations of an MMP-1 $ THP complex was performed. To examine the role of the linker region for MMP-1 function simulations with linker regions from MT1-MMP/MMP-14 and MMP-13 replacing the MMP-1 linker region were performed. The MD studies were in good agreement with the experimental observation that in the MMP-1 $ THP X-ray crystallographic structure MMP-1 is in a “ closed ” conformation. MD revealed that the interactions of the THP with the both the CAT and HPX domains of MMP-1 are dynamic in nature, and the linker region of MMP-1 in ﬂ uences the interactions and dynamics of both the CAT and HPX domains and collagen binding to MMP-1.


Introduction
One of the primary components of the extracellular matrix (ECM) is collagen, which is also the most abundant protein in mammals. 1 There are at least 29 different types of collagens that occur in vertebrates.The collagen molecule consists of three polypeptide strands (a chains) that organize themselves in a ropelike triple-helix conformation, stabilized by inter-chain hydrogen bonding.The degradation of collagen is a key process in normal development and homeostasis; however, unbalanced collagenolysis contributes to numerous pathologies such as cancer, arthritis, and cardiovascular and neurodegenerative diseases.Amongst the enzymes capable of collagen catabolism in vertebrates are matrix metalloproteinases (MMPs).][4][5] MMP-1 degrades interstitial (types I-III) collagen into 3/4 and 1/4 fragments. 6,7The three-dimensional X-ray crystallographic structure of catalytically inactive (E200A) MMP-1 complexed with a collagen-model triple-helical peptide (THP) has been solved by Manka et al. (Fig. 1). 8MMP-1 in this structure consists of the N-terminal catalytic (CAT) domain, the C-terminal hemopexin-like (HPX) domain, and the linker region connecting the CAT and HPX domains.The THP, with three strands designated as leading (L), middle (M), and trailing (T), is approximately 115 A in length and makes extensive interaction with both the CAT and HPX domains. 8In concert with the X-ray crystallographic "snapshot" of MMP-1$THP, dynamic information about MMP-1 interaction with triple-helices was obtained through a series of NMR spectroscopic structures and molecular docking calculations. 9olecular dynamics (MD) simulations have been applied to study the MMP family of enzymes.Diaz and coworkers observed a conformational shi from a closed X-ray crystallographic MMP-2 structure to an elongated structure using atomistic MD simulations on a 100 ns time scale. 10MMP-2 can adopt extended conformations in solution prior to collagen hydrolysis. 11MD has provided insight into the degradation of type I collagen brils by MMP-8, 12 but limited efforts have been made to study MMP-1.Presently, the largest collection of experimental data for MMP-catalyzed collagenolysis has been obtained using MMP-1, 8,9,[13][14][15] yet there is still much to be learned as to how conformational exibility and dynamics inuences MMP-1 structure-function relationships, and specically to provide insight on the effects of dynamics on the interactions between the MMP-1 CAT and HPX domains and with collagen triple-helices.In addition, it is important to understand how the composition and the exibility of the linker region inuence the interactions between the MMP-1 CAT and HPX domains and with collagen.In order to provide insight into MMP-1-catalyzed collagenolysis, we herein performed a set of long-range atomistic (AT) MD simulations on an MMP-1$THP complex.Effects of the linker region on inter-domain interactions and interactions with the substrate were studied by MD simulations of in silico engineered MMP-1 with the linker of MMP-14/MT1-MMP or MMP-13.

Initial structure preparation
The coordinates of the wild type MMP-1 bound to a THP model of the type II collagen MMP cleavage site were obtained from the Protein Data Bank 18 (PDB 4AUO 8 ).SwissPDBViewer 19 was used for adding missing atoms and selecting one from the alternative side chain orientations. 19The active form of MMP-1 was made by substituting the A200 with E200 using the Modeller 20 program.The linker of MMP-14 and MMP-13 were modelled into the linker region of MMP-1 by using the Modeller 20 program.The numbering of the THP in the MMP-1$THP complex and subsequence simulations was assigned as 763-795, based on the sequence numbering within the triple-helical region of type II collagen, instead of 963-995 as in the X-ray crystallographic structure.

Molecular dynamics simulations
In order to explore the dynamic properties of MMP-1, we performed an extensive set of MD simulations for 300 ns (wild type MMP-1) and 100 ns for the modied linker systems (Table S1 †) by using Gromacs 4.5.5 package [21][22][23] with GROMOS96 43a1 (ref.24) force eld.The protonation states of His residues in the protein molecule were assigned based upon the optimal hydrogen bonding conformation performed in Gromacs using pdb2gmx.However H149, H164, H199, H203, and H209 were protonated at their delta and H177 at the epsilon position according to their local environment in the vicinity of Zn 2+ .The Zn 2+ and Ca 2+ ions were restrained in MD simulation according to their X-ray crystallographic structure distance using harmonic potential.In vacuo energy minimization was performed to remove steric clashes in the crystal structure rst by using the steepest descent 25 and then by using conjugate gradient 26 until the maximum force was found smaller than 100 kJ mol À1 nm À1 .The editconf command was used to dene the dimension of the cubical box and the protein molecule was placed in the box.The periodic boundary conditions were then applied to treat all the parts of the system equally both at its interior and edges.The box size was set to ensure a distance of at least 1.0 nm between the protein and the box boundaries.The energy minimized protein structure was then solvated by using Single Point Charge (SPC) 27 water model in the cubical simulation box by using Genbox command.The system was neutralized by adding Cl À to MMP-1 and the linker-modied enzymes (Table S1 †).In order to relax the solvent molecule and remove constrains from the entire system, the energy minimization of the whole system was performed using rst the steepest descent and then the conjugate gradient algorithm until the maximum force was found smaller than 100 kJ mol À1 nm À1 .The energy minimized structure was then subjected to position restrain dynamics for 50 ps.The simulation was performed in NVT ensemble (constant number of particles, volume, and temperature) 28 at constant temperature of 300 K with time step of 0.002 ps.The Berendsen temperature coupling and Parrinello-Rahman pressure coupling were used to keep the system at 300 K and 1 bar pressure during the simulation procedure.The productive MD was carried out using NVT ensemble and the initial velocities for MD simulation were drawn from Maxwell velocity distribution at 300 K.The MD was performed with an integration time step of 0.002 ps.The Particle Mesh Ewald (PME) 29 method was used for electrostatic interactions with Coulomb cut off of 1.0 nm, Fourier spacing of 0.135 nm tolerance of 1 Â 10 À5 and an interpolation order of 4. The Lennard Jones potential was employed for the treatment of van der Waals interaction with cut off distance set to 1.4 nm.
Fig. 1 (A) The 3D structure of human MMP-1 (E200A) complexed with a THP (PDB 4AUO 8 ) drawn using UCSF Chimera. 16MMP-1 (displayed in silhouette round ribbon) consists of the CAT domain (orange), interdomain linker (red), and HPX domain (grey).The L, M, and T strands of the THP are shown in tube representation in cyan, green, and red, respectively.The zinc and calcium ions bound to the enzyme are shown in spherical representation in blue and purple, respectively.(B) 2D topology diagram of (E200A) MMP-1 made using Pro-origami tool using STRIDE algorithm for secondary structure prediction. 17 The LINCS algorithm 30 was utilized to keep all the covalent bonds involving hydrogen atoms rigid.

Analysis of molecular dynamics simulations
The analyses of the trajectories obtained from the simulations were performed using tools from the Gromacs soware package.The Root Mean Square Deviation (RMSD) of Ca atoms of the protein with respect to minimized crystal structure, Root Mean Square Fluctuations (RMSF), electrostatic interactions, hydrogen bonding, Solvent Accessible Surface Area (SASA), and cluster analysis were performed.The visualization of MD trajectories and the structures were performed using VMD soware. 31The Bio3D 32 package in R was used to produce principal component (PC) analysis and domain cross correlation.Center of mass was calculated using Ca atoms of the CAT and HPX domains over simulation time using g_dist in Gromacs and also CALCOM 33 to verify the results obtained from the g_dist in Gromacs.

Dynamic cross correlation analysis
Correlated and collective atomistic motions play an important role in the functionality of dynamic biomolecular systems. 34The Ca atoms of the protein and THP were used to create a cross correlation C ij matrix using MD trajectory.The cross correlation between the ith and jth an atom were represented by a C ij matrix and was extracted by the following equation: The Dr i and Dr j are the displacement vectors corresponding to ith and jth atom and the angle brackets denote an ensemble average.The C ij matrix ranges from +1 to À1, where the positive value represented the correlated motion and the negative values represented the anticorrelated motion.

Conformational dynamics of MMP-1$THP complex
The overall stability of the MMP-1$THP complex and changes associated within the internal structure and geometry of the individual CAT and HPX domains and linker region were assessed by computing the RMSD of the backbone Ca atoms with respect to the minimized X-ray crystallographic structure (PDB 4AUO).MMP-1$THP equilibrated aer 10 ns with an average RMSD value of 4.7 A (Fig. 2, S1A, and S1B, Table S2 †).This RMSD is comparable to the RMSD of 4.2 A of the collapsed (closed) form of MMP-1 in complex with the THP obtained from NMR studies. 9Three different runs of MMP-1 (Fig. S1B †) were performed in order to enhance the conformation sampling and avoid statistical errors, with an average value of 5.1 A obtained (Table S3 †).The multiple MD simulations were performed in order to evaluate the effect of the statistical noise on the quality of and to test the stability of the simulations.There is a discussion in the MD community about the impact of statistical errors on the quality of the MD simulations, and one of alternatives of single simulation is to run multiple MDs from the same starting structure with different initial velocities which allow evaluation of the quality of the single trajectories and to minimize the force-eld related artifacts.The four simulations of MMP-1 show good consistence and convergence of the individual trajectories, as indicated by the RMSD plots (Fig. S1B †).Full length MMP-2 in a compact structure (PDB 1CK7) exhibited an average RMSD value of 4 A during the rst 70 ns of simulations performed by Diaz and co-workers. 10The MD simulation of MMP-2$fTHP-5 performed by Diaz and coworkers showed on average RMSD of $6 A. 35 The MMP-1 CAT domain equilibrated at $10 ns and was stable throughout the simulation with RMSD less than 2 A (Fig. S1 †).The HPX domain showed a dri at $60 ns with an average value of 2.6 A and then stabilized for the entire length of the simulation (Fig. S1 †).The linker region was also stable with an RMSD of 2 A and showed equilibration at $10 ns (Fig. S1 †).The linker region underwent limited structural deviation indicating that the inter-domain arrangement in MMP-1 in solvent is maintained close to the X-ray crystallographic structure throughout the simulation.The RMSD of the CAT and HPX domains and the linker of MMP-1 were comparable to the MD results from the simulation of the CAT domain (2.9 A), HPX domain (2.4 A), and linker region (2.1 A) of MMP-2 complexed with THPs. 36The RMSD data suggested that the HPX domain shows greater structural deviation in comparison to the CAT domain.The simulations of MMP-2 also showed higher structural deviation of the HPX domain (1.7 A) in comparison to CAT domain (1.1 A) aer 200 ns. 11The CAT and HPX domains in MMP-2$fTHP-5 showed RMSD values of $2 and $4 A, respectively, and the HPX domain showed greater structural deviation in contrast to CAT domain. 35he THP exhibited a high RMSD value (7 A) relative to components of MMP-1 (Fig. S1 †) indicating a signicant structural change in comparison to the X-ray crystallographic structure.The THPs in the MMP-2 complex also exhibited a higher RMSD (on average 4.7 A). 36 In order to evaluate the compressing effect of the enzyme on the triple-helix dynamics, Fig. 2 The RMSD of all Ca atoms of the MMP-1$THP complex.The RMSD of MMP-1 is compared with MMP-1 with the linker of MMP-14 (MMP-1/MMP-14) and MMP-1 with the linker of MMP-13 (MMP-1/ MMP-13).
free THP in water was simulated.The average RMSD value of the THP in water was 22 A with respect to 7 A in the MMP-1$THP complex for 50 ns simulation (Fig. S1 †), which implied a restraining effect of the enzyme on the THP and also highlighted the extent of conformational exibility and dynamics of the THP in solution.Comparison of the averaged structures of the THP in water (based upon cluster analysis of the most populated cluster) and in the enzyme$substrate complex indicated considerable conformation changes (Fig. 3), although the triple-helical nature of the THP in water was maintained by consistent interchain hydrogen bonding.Enhanced conformational exibility of THPs during MD has been reported previously 37,38 as well the dynamic nature of the individual strands and their associated structures. 49he exibility of MMP-1 and THP residues was assessed using RMSF analysis (Fig. 4A and S2A †).Average RMSF values were 1.14 A for the CAT domain, 2.15 A for the linker, 1.40 A for the HPX domain, and 2.0 A for the THP.The linker had uctuations which peaked at 3.1 A while the HPX domain displayed greater uctuations relative to the CAT domain.The RMSF value of linker region in the compact conformation of MMP-2 was approximately 2.4 A. 39 The RMSF values of residues belonging to the HPX domain of MMP-2 also exhibited greater uctuations in contrast to its CAT domain and linker. 11,36The loop region between parallel b-strands b2, b3, and b4 presented high uctuations in the MMP-1 CAT domain.The aC of the CAT domain and the loop connecting the linker to b6 of the HPX domain showed increased exibility.Increased uctuations in the HPX domain was seen in the region encompassing residues of b17 and the loop between b17 and b18.The basal level uctuations for MMP-1 were considered to be 1.4A (based upon the mean value), where 43% of the residues showed uctuations greater than 1.4 A and $56.9% of the residues showed uctuations less than 1.4 A (Table S2

†).
The highest peak in the RMSF plot was from the N-and Ctermini of the THP (Fig. 4A).The RMSF prole of the scissile bond (G775-L776) also showed high exibility for the M and T strands with respect to the L strand (Fig. S2A †).In the MD simulations of MMP-2 the N-and C-termini of the THP bound to MMP-2 also exhibited high RMSF in the range of 8 to 16 A, respectively. 36 analysis can provide information on the overall dynamics of the THP in water.PC1 and PC2 accounted for $90% of the overall motion of the THP in solvent (Fig. S2B †).The maintenance of triple-helical structure and PC analysis of the THP in water shows good agreement with the uorogenic THP simulation in aqueous solution where the rst PC accounts for the twist motion of the triple-helix and the triple-helix is maintained throughout 50 ns trajectory. 40The projection of PC1 on the THP indicated bent or twist conformation as has been described by cluster analysis.The dynamics cross correlation analysis of the THP chains showed signicant correlated motion through the 50 ns trajectory (Fig. S2C †).The extent of both positive and negative motion were in accordance with the maintenance of triple-helical structure during simulation.
The L strand of the THP approached closest to the active site in the simulation and exhibited the largest bending out of all of the THP strands (Fig. S3 †).Experimental studies on MMP-1 (ref.9, 14 and 15) suggested that the active site of MMP-1 could not accommodate the 15 A diameter of the collagen triple-helix.Therefore, the sequential hydrolysis of single collagen strands occurs through the binding of the triple-helix to the HPX domain and allowing inter-domain conformational exibility through the linker to facilitate the reorientation of CAT domain to position itself to perform catalysis.The reaction mechanism of collagen hydrolysis by MMPs requires the carbonyl group of the scissile peptide bond to coordinate with the catalytic Zn 2+ in active site. 39,41The scissile bond between residues G775 and Fig. 3 Superposition of the THP in water versus the THP in the MMP-1$THP complex.The structure of the most populated cluster of THP in water (brown) was aligned against the structure obtained from the most populated cluster of THP in the MMP-1$THP complex (cyan).L776 of the L strand is located 8.8 A from the catalytic Zn 2+ in the CAT domain; therefore, the X-ray crystallographic structure of MMP-1 bound to the THP is unproductive due to absence of the scissile bond near the S 1 -S 0 1 subsites in the CAT domain.In the MD simulations, the bond distance decreased on average to 6.7 A (Fig. S4 †) but still does not represent the active complex.

Triple-helix interactions with the CAT and HPX domains
It has been demonstrated experimentally that specic residues present in blades 1 and 2 of the HPX domain interact with the collagen triple-helix. 8,9,13,14In the present simulation specic residues within blade 1 (residues 250-300) belonging to b6-b9 (Fig. 1B) interacted with the THP (Fig. S5 †).New sets of interactions emerged between the L strand of the THP and the HPX domain.The backbone of L295 makes hydrogen bonds with the side chain of R780 with an average distance of 3.5 A. The side chain of N296 participated in hydrogen bonding with the backbone of G781 and hydrophobic interaction with the aliphatic side chain of R780.The guanidino group of R780 had a close interaction with the carboxylic group of E294 (average distance of 4.5 A) with a weak electrostatic interaction between the residues.
The X-ray crystallographic structure revealed a hydrophobic cluster formed by V300 and F301 side chains with V783 of the L THP strand. 8This interaction was lost in the MD simulation and a new hydrophobic interaction between the side chains of P303 and I782 was established (Fig. S6 †).A hydrogen bond also emerged between the side chain of Q335 and G784 (L strand) (Fig. S6 †).R780 to G784 are located in the THP strands which are most bent during the simulation.R780 makes extensive interactions with the HPX domain and could be a reason why the THP is so bent during the simulation.The M strand of the THP made most of the interactions with the HPX domain in similar fashion to the X-ray crystallographic structure; nonetheless, new interactions emerged from the simulation studies, specically involving T270 and N296 (Fig. S7 †).R272 made hydrogen bonds with the backbone and side chain of O786 (M strand) and R789 (M strand), respectively, as described in the Xray crystallographic structure.The aliphatic region of R272 was involved in formation of a hydrophobic cluster with the side chains of R789 and L785 of the M THP stand (Fig. S8 †).
We previously demonstrated experimentally that the MMP-1 HPX domain binds to the THP with 1 : 1 stoichiometry. 9This suggested that the HPX domain binds to more than one THP chain.The MMP-1 X-ray crystallographic and NMR structures 8,9 showed the interaction of L and M strands of the THP with residues of blades 1 and 2 of the HPX domain.Our MD simulation also showed the existence of interactions between the HPX domain and two THP strands (L and M).
The CAT domain mainly interacts with the L strand of the THP (Table S4 †).The X-ray crystallographic structure indicated that the Y221 backbone formed a hydrogen bond with the side chain of Q779 (L strand); however, the simulation results revealed that the side chain of Q779 (L strand) moves away with an average distance of 5.5 A from the backbone NH group of Y221 and makes a close interaction in the S 1 subsite of the CAT domain (Fig. S9 †).The MD studies conrm the presence of the Q779 side chain (L strand) in the S 1 subsite with an average distance of 4.7 A (Fig. S10 †) further indicating the absence of the scissile bond near the active site.Our MD shows no signicant interactions been made by a residue of the CAT domain with residues in vicinity of scissile bond, thus con-rming the unproductive orientation in the X-ray crystallographic structure.
Flexibility and domain interactions in MMP-1 with linker regions from MMP-14 or MMP-13 The linker regions in MMPs have been proposed to play important roles for the mutual orientation of the domains and their interactions with collagen. 9,14,15In order to explore the effects of the nature of the MMP-1 linker region (both the length of the linker and its amino acid sequence) on modulating domain interactions, it was exchanged with the linker region from MMP-14 (which contains 35 residues compared with 17 residues in MMP-1) or MMP-13 (which consists of the same number of residues as the MMP-1 linker but with a different sequence). 42The trajectory of MMP-1 with in silico MMP-14 linker (MMP-1/MMP-14) showed that the system equilibrated at $20 ns (Fig. S11 †).The average RMSD value of MMP-1/MMP-14 was 5.6 A compared to 4.7 A for MMP-1 (Fig. 2).Comparison of the RMSD of the CAT and HPX domains of MMP-1/MMP-14 with MMP-1 indicated that the CAT domain of MMP-1/MMP-14 showed slightly higher structural deviation in comparison to MMP-1 (Fig. S12 †).The average RMSD values of the linker region are 5.6 A for MMP-1/MMP-14 and 2.0 A for MMP-1 (Fig. S13 †) indicating greater structural deviation in the former case.These differences could have signicant impact on interactions between the CAT and HPX domains and would affect the conformational dynamics of both domains.
The basal level uctuation for MMP-1 was considered to be 1.4 A. In MMP-1/MMP-14 57% of the residues showed uctuations greater than 1.4 A (Table S2 †) indicating an overall increase of 14% higher uctuations in MMP-1/MMP-14 compared to the RMSF of MMP-1 (Fig. S14 †).This increase could be attributed to the larger size of the MMP-14 linker (35 amino acids) compared to the 17 amino acid residue linker of MMP-1.The RMSFs for both domains of MMP-1/MMP-14 showed on average >10% overall increase in comparison to MMP-1 (Table S5 †).The THP complexed to MMP-1/MMP-14 showed 20% increased uctuations with respect to the THP complexed to MMP-1 (Table S5 †).The overall effect of the MMP-14 linker in MMP-1 was an increase in the exibility of the enzyme$substrate complex.Residues 171-180 encompassing the loop between b4 and b5 exhibited increased uctuations in MMP-1/MMP-14.These residues are in vicinity of the structural Zn 2+ in the CAT domain.Residues 188-191 encompassing the loop between b5 and aB also showed increased uctuations in MMP-1/MMP-14.The adjacent aB helix contains H199, E200, and H203, which are involved in coordination with the catalytic Zn 2+ in the CAT domain.The residues of the loop between ahelices aB and aC showed increased exibility.This loop contains H209, which is coordinated to the catalytic Zn 2+ .There was signicantly more uctuation in the HPX domain compared to CAT domain of MMP-1/MMP-14 (Fig. S14 †).
The linker region in MMP-13 is the same size as the linker in MMP-1, but with a different sequence. 42The RMSD prole of MMP-1 with the linker of MMP-13 (MMP-1/MMP-13) showed that the system equilibrated at $20 ns (Fig. 2) and indicated that MMP-1/MMP-13 had greater structural deviation with average value of 5.3 A in comparison to MMP-1 (4.7 A).The RMSD prole of the linker in MMP-1/MMP-13 showed a similar trend to the MMP-1 linker, but exhibited higher exibility (Fig. S15 †).The RMSF plot of MMP-1/MMP-13 showed overall increased uctuations in comparison to MMP-1 (Fig. S16 †).In MMP-1/MMP-13 68% of the residues exhibited uctuations greater than 1.4 A (Table S2 †) indicating an overall increase of 25% higher uctuations compared to the RMSF of MMP-1.The RMSF of the two domains of MMP-1/MMP-13, in contrast to MMP-1, showed that the CAT domain of MMP-1/MMP-13 contains on average 64% residues with RMSF > 1.4% with respect to 31.6% in MMP-1.The HPX domain of MMP-1/MMP-13 contained 58.5% of residues with uctuations >1.4% as compared to 46.8% in MMP-1.Comparison of the RMSF of the linker region residues showed an increase in comparison to MMP-1 that is slightly higher than in the linker of MMP-1/MMP-14 (Fig. 4B).An increase in the exibility of residues in the CAT domain of MMP-1/MMP-13 was seen in the loop connecting aB and aC.The aC is directly connected to the linker region of MMP-1/MMP-13.
In order to understand how the inter-domain distance between CAT and HPX domain changes as a function of time we computed the distance between centers of mass of the CAT and HPX domains.The average inter-domain distances for MMP-1, MMP-1/MMP-14, and MMP-1/MMP-13 were 37.8, 37.0, and 37.7 A, respectively, and remained relatively stable throughout the simulation (Fig. S17 †).The inter-domain distance of MMP-1/ MMP-14 did show a decrease aer 80 ns simulation (Fig. S17 †).The center of mass between the CAT and HPX domain in MMP-2 also showed similar trends with an average distance of 39.6 A and remained stable throughout the entire length of simulation. 11,36The average distance between the scissile bond of the THP L strand and the CAT domain (catalytic Zn 2+ ) was 6.7 A in MMP-1, 8.4 A in MMP-1/MMP-14, and 8.3 A in MMP-1/MMP-13 (Fig. S18 and S19 †), illustrating that changing the linker does not lead to a more productive conformation, but rather the opposite.
The backbone of Y221 in MMP-1 formed hydrogen bonds with the backbone of R780 of the L THP strand (Table S4 †).However, in MMP-1/MMP-14 this interaction was not maintained during the simulation (Fig. S22 †).The side chain of Q779 of the THP L strand no longer accessed the active site of MMP-1/MMP-14 and was on average 10.3A away, while in MMP-1 it was 4.7 A from the active site.The backbone of S220 formed hydrogen bonds with the side chain of Q779 aer 40 ns simulation in MMP-1 (Table S4 †), while there were no hydrogen bonds formed between the S220 backbone and the side chain of Q779 in MMP-1/MMP-14 (Fig. S23 †).R780 of the THP L strand interacts with the MMP-1 HPX domain, while all of the interactions of R780 seem to be very unstable in the MMP-1/MMP-14 complex throughout the entire length of the simulation (Fig. S24 †).R272 of the HPX domain was extensively involved in hydrogen bonding with the L785, O786, and R789 residues of the THP M strand in MMP-1.In MMP-1/MMP-14 the side chain of R272 makes a stable interaction with the backbone of O786 for 40 ns and disappears as the simulation evolved over time (Fig. S25 †).
The change in atomistic interactions in MMP-1/MMP-14 in comparison to MMP-1 pinpoints the role of the linker in modulating the interactions between the enzyme and substrate.The secondary structure of the linker region of MMP-1 appears relatively stable as a function of simulation time (Fig. S26 †).However, the linker of MMP-1/MMP-14 showed changes in secondary structure during the simulation (Fig. S27 †).
In MMP-1/MMP-13, the Q779 side chain of the THP L strand accessed the active site S 1 pocket with an average distance of 3.3 A compared to 4.7 A in MMP-1.The backbone of Y221 and P219 in MMP-1/MMP-13 made a hydrogen bond with R780 of the THP L strand in a similar manner as in MMP-1.The side chain of Q167 also made hydrogen bonds with the O771 side chain of the L strand as it did in MMP-1 (Fig. S28 †).Residues of the HPX domain such as N296, T270, and R272 showed a similar pattern of interaction as MMP-1/MMP-14 (Fig. S29 †).These results indicated that overall the interactions of the CAT domain with the THP in MMP-1/MMP-13 is closer to MMP-1, while the HPX domain interactions of MMP-1/MMP-13 showed a pattern which is closer to MMP-1/MMP-14 than MMP-1.The structures obtained from the most populated clusters from cluster analysis of MMP-1, MMP-1/MMP-13, and MMP-1/MMP-14 showed signicant differences in orientation and binding of the THP to both the CAT and HPX domains (Fig. 5 and 6).The linker clearly modulates important interactions in both the CAT and HPX domains.Cross correlation analysis of MMP-1/MMP-14 and MMP-1/ MMP-13 was subsequently performed (Fig. 8 and 9, crosscorrelation maps; Fig. S30, † the correlated elements from the protein structures).The terminal residues of the linker region of MMP-1/MMP-14 showed correlation with the loop region of the C-terminus (residues 439-447) of the HPX domain.Residues 422-427 of b22 of the HPX domain also showed correlation towards the residues of the linker region.The residues in the loop connecting the C-terminus of the HPX domain showed positive correlation toward the linker region residues.Residues 409-413 of b20 showed correlation towards residues 262-267  located in vicinity of the linker region in the HPX domain.One important observation was the low degree of correlated motions observed between the THP and the CAT and HPX domains of MMP-1/MMP-14 in comparison to MMP-1.Interestingly, there was signicant positive correlation between the residues of the linker region and the HPX domain in MMP-1/MMP-14.Overall, MMP-1 has more correlated and anti-correlated motion than MMP-1/MMP-14.
The terminal residues of the MMP-1/MMP-13 linker region showed correlated motion towards the C-terminal residues (436-444) and b21 of the HPX domain.The residues of the loop region between b8-b9 (288-296) showed correlation towards the C-terminal residues (436-444) of the HPX domain.Residues of the linker region of MMP-1/MMP-13 and MMP-1/MMP-14 have positive correlation towards the HPX domain, which was not observed with the natural linker of MMP-1.The greater extent of positive and negative correlation in MMP-1/MMP-13 in contrast to MMP-1 indicated that even changes in the sequence of the linker produces signicant differences in the motion of residues.

Conclusions
MD simulations of the X-ray crystallographic structure of MMP-1 (ref.8) revealed that conformational changes exercise an important role and inuence the interactions between the enzyme HPX and CAT domains and revealed the existence of extensive correlated motions.The interactions between the THP chains and the enzyme also have a exible nature.Importantly, we conrm prior NMR experimental results 9 that the X-ray crystallographic structure does not represent the functional enzyme$substrate (E$S) complex.Although we used this nonproductive enzyme-substrate complex as a starting point for the MD simulations, as opposed to the productive complex reported from NMR spectroscopic studies, it is important to note that the X-ray crystallographic structure of MMP-1$THP is the only structure that denes the specic molecular interactions between the enzyme and substrate, as the NMR-derived structure is based on docking of residues.The MD simulation results are relevant to MMP-1 catalyzed collagenolysis, as the X-ray crystallographic and NMR structures of MMP-1$THP agree on the majority of THP interactions with the HPX domain and agree on the "closed" conformation of MMP-1 during collagenolysis.The signicant point of departure in the two structures is the position of the THP with regards to the CAT domain, where the NMR-derived structure represents a productive complex while the X-ray crystallography-derived structure does not.The effects of these differences on MMP-1 dynamics and correlations can be explored in subsequent studies using MD simulations.
The correlated motions observed here between the residues from the CAT domain and locally destabilized THP region would be important for the formation of the productive E$S complex.The correlated motions would also support a more favourable orientation of the enzyme electrostatic environment that would facilitate the catalyzed hydrolysis reaction in the active site (e.g., stabilizing the transition state).The simulations indicate the importance of implementing multiple structures from MD trajectories for reaction path calculations, as opposed to single structure based-calculations.The effect of the conformational exibility of the E$S complex on the enzyme reaction path has already been demonstrated in number of mechanistic studies, such as cyclooxygenase-1. 48he linker plays a key role in modulating the interactions between the domains and formation of the active complex.The MD simulations with exchanged linker regions from MMP-14 and MMP-13 reveal the important role of the linker region to inuence overall enzyme exibility, the pattern of the correlated  motions between the HPX and CAT domains, and the interactions between the triple-helical substrate and the enzyme.Prior experimental studies examined the effects of MMP linker exchange on collagenolysis.A chimeric MMP-8 whose linker region (17 residues) was replaced with the corresponding MMP-3 sequence (26 residues) lost activity towards collagen. 43In similar fashion, MMP-1/MMP-3 chimeras possessing the MMP-3 linker were not active towards collagen. 44,45The linker has been proposed to be critical for proper orientation of the CAT and HPX domains. 9,46,47The present study furthers the role of the linker, as it was found to facilitate correlative interactions between MMP domains and MMP domains and triple-helices.Overall, examination of MMP-1$THP demonstrates the potential of MD simulations to provide additional insight into experimentally-derived data.
Fig.1(A) The 3D structure of human MMP-1 (E200A) complexed with a THP (PDB 4AUO 8 ) drawn using UCSF Chimera.16MMP-1 (displayed in silhouette round ribbon) consists of the CAT domain (orange), interdomain linker (red), and HPX domain (grey).The L, M, and T strands of the THP are shown in tube representation in cyan, green, and red, respectively.The zinc and calcium ions bound to the enzyme are shown in spherical representation in blue and purple, respectively.(B) 2D topology diagram of (E200A) MMP-1 made using Pro-origami tool using STRIDE algorithm for secondary structure prediction.17The color gradient of blue and red are used for the N-and C-termini of the protein.The b-sheets are represented numerically and the a-helices alphabetically.

Fig. 8
Fig. 8 Dynamic cross correlation analysis of MMP-1/MMP-14$THP.Intensities are indicated by the bar on the right.

Fig. 9
Fig. 9 Dynamic cross correlation analysis of MMP-1/MMP-13$THP.Intensities are indicated by the bar on the right.