proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Molecular dynamics simulations of theactive matrix metalloproteinase-2:Positioning of the N-terminal fragmentand binding of a small peptide substrateNatalia Dıaz* and Dimas Suarez

Departamento de Quımica Fısica y Analıtica, Universidad de Oviedo, Oviedo (Asturias), Spain

INTRODUCTION

Matrix metalloproteinases (MMPs) are a family of structurally related,

zinc- and calcium-containing enzymes that catalyze the breakdown of connec-

tive tissue. Accordingly, they play a central role in all the physiological pro-

cesses requiring tissue turnover and remodelling, but their expression is also

known to increase in various inflammatory, malignant, and degenerative dis-

eases. This has led to considerable efforts in the development of MMP inhibi-

tors that would be applied during the treatment of these pathologies.1,2

However, the toxicity demonstrated in clinical trials by some of these inhibi-

tors, which most likely results from a nonspecific inhibition, clearly stresses

the need for more selective compounds capable of discriminating among the

different members of this important protease family.3,4

The MMP-2 enzyme, also termed gelatinase A, plays a key role in angio-

genesis thanks to its ability to hydrolyze collagen type IV, which is the main

component of the basement membrane, as well as interstitial collagens like

type I.5,6 In addition, the expression of MMP-2 has been demonstrated in

many different human tumors and inflammatory processes.7–9 The tridimen-

sional structures currently available for MMP-2 correspond to the latent

pro-enzyme (PDB codes: 1CK7, 1GXD, and 1EAK), in which an N-terminal

pro-peptide (Pro31-Asn109; 1CK7 numbering) blocks the access to the active

site cleft in the catalytic domain.10,11 In addition, X-ray crystallography and

nuclear magnetic resonance (NMR) methods have been employed to analyze

the structure of the isolated catalytic domain of the enzyme bound to

hydroxamic acid inhibitors, which interact with the catalytic zinc ion (Zn1)

located in the active site.12,13 However, the structural and dynamical details

of the interaction between the active MMP-2 enzyme and their relevant pep-

tide substrates, like the collagen triple helix, are largely unknown. This infor-

mation would be mostly relevant to characterize the reaction mechanism

catalyzed by the enzyme and to elucidate the regions that can be exploited in

the rational design of more selective inhibitors.

The crystal structures of several MMP enzymes bound to different types of

peptidomimetic inhibitors suggest that peptide substrates would align in the

active site groove by establishing H-bond contacts with several main-chain

The Supplementary Material referred to in this article can be found online at http://www.interscience.wiley.

com/jpages/0887-3585/suppmat/

Grant sponsor: Spanish MEC; Grant number: CTQ2004-06309.

*Correspondence to: Natalia Dıaz, Departamento de Quımica Fısica y Analıtica, Universidad de Oviedo, C/ Julian

Claverıa, 8. 33006, Oviedo (Asturias), Spain. E-mail: diazfnatalia@uniovi.es

Received 6 September 2007; Revised 24 October 2007; Accepted 25 October 2007

Published online 10 January 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.21894

ABSTRACT

Herein we use different computational

methods to study the structure and

energetic stability of the catalytic do-

main of the active MMP-2 enzyme con-

sidering two different orientations of its

N-terminal coil. The first orientation is

largely solvent accessible and corre-

sponds to that observed in the 1CK7

crystal structure of the proenzyme. In

the second orientation, the N-terminal

coil is packed against the X-loop and

the a3-helix of the MMP-2 enzyme like-

wise in the so-called ‘‘superactivated’’

form of other MMPs. Binding to the

MMP-2 catalytic domain of a short pep-

tide substrate, which mimics the

sequence of the a1 chain of collagen

type I, is also examined considering

again the two configurations of the N-

terminal coil. All these MMP-2 models

are subject to 20 ns molecular dynamics

(MD) simulations followed by MM-

PBSA (Molecular Mechanics Poisson-

Boltzmann Surface Area) calculations.

The positioning of the N-terminal coil

in the ‘‘superactivated’’ form is found to

be energetically favored for the MMP-2

enzyme. Moreover, this configuration of

the N-terminal moiety can facilitate the

binding of peptide substrates. Globally,

the results obtained in this study could

be relevant for the structural-based

design of specific MMP inhibitors.

Proteins 2008; 72:50–61.VVC 2008 Wiley-Liss, Inc.

Key words: enzyme catalysis; hydrolysis;

metalloenzymes; molecular modelling;

structural biology.

50 PROTEINS VVC 2008 WILEY-LISS, INC.

groups located along the b4-strand and in the X-loop of

the catalytic domain, adopting thus a b-sheet structure

(see Scheme 1). Simultaneously, the substrate side chains

would interact with the so-called S3–S03 sites of the cata-

lytic domain.14 According to substrate specificity analy-

sis, two well-defined hydrophobic sites, the S3 and S01pockets, are best suited to accommodate proline residues

and long hydrophobic side chains from the ligands,

respectively.14 Interestingly, previous molecular dynamics

(MD) simulations have shown that the accessibility of

the main binding sites is related to the metal ion content

(Zn(II) and Ca(II)) of the catalytic domain.15 The MD

analyses, performed for the MMP-2 enzyme, point out

that the main binding groups are all accessible only when

two zinc and two calcium ions are bound to the catalytic

domain as observed in the 1CK7 crystal structure.

Besides the structure of the catalytic groove in the

active form of the MMPs, the length and positioning of

the N-terminal coil is also relevant to the proteolytic

activity of the enzyme, a phenomenon that has been

termed ‘‘superactivation’’ in the past.16 Thus, it has been

reported that, in vitro, different activation protocols of

the proenzymes result in different N-terminal residues in

the catalytic domain and in a slightly different hydrolytic

activity.16–18 Subsequent crystallographic studies per-

formed for MMP-1 confirmed that the ‘‘superactivated’’

enzyme displays the N-terminal coil of the catalytic do-

main packed against a concave hydrophobic surface

made up by the a3-helix and part of the long X-loop.The interaction between the N-terminal ammonium

group and the carboxylate side chain of a strictly con-

served aspartic acid from the a3-helix contributes to this

packing of the N-terminal coil.19

According to protein sequence data, the first residue in

the catalytic domain of the active MMP-2 enzyme is

Tyr110.20 However, the two tridimensional structures

reported for this domain begin at Arg115 or Met109, and

both show a quite disordered N-terminus. Thus, no

structural information is available for the MMP-2

enzyme in a conformation similar to that observed in the

‘‘superactivated’’ forms of other MMPs. In this work, we

built computational models, readily comparable with

each other, of the ‘‘N-terminal disordered’’ and ‘‘superac-

Scheme 1Typical MMP-2/substrate contacts. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]

Figure 1Ribbon model of the catalytic domain of the MMP-2 enzyme and a molecular surface representation of the hydrophobic groove flanked by the a3-helix and the X-loopshowing the N-UP and N-DOWN orientations adopted by the N-terminal fragment (in sticks). The zinc (in magenta) and calcium (in green) ions are also displayed

as spheres.

N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 51

tivated’’ forms of the MMP-2 enzyme starting at Tyr110.

The resulting configurations are denoted arbitrarily as

N-UP and N-DOWN, respectively (see Fig. 1). We exam-

ined first the likely conformations of MMP-2 in the two

different states of the N-terminal fragment by computing

long MD trajectories (20 ns) for the fully solvated

enzyme. From the MD simulations, we analyzed in detail

the influence of the position of the N-terminal coil on

the other structural elements and on the accessibility of

the important binding sites. Furthermore, the relative sta-

bility of the different MMP-2 configurations was assessed

on the basis of Molecular-Mechanics Poisson-Boltzmann

Surface-Area (MM-PBSA) calculations, which were previ-

ously validated by carrying out quantum chemical calcu-

lations on model systems. We also set up computational

models of the MMP-2 enzyme bound to a small peptide

substrate (Ace-Gly-Pro-Gln-Gly�Ile-Ala-Gly-Gln-Nme),

which mimics the sequence of the a1 chain of collagen

type I that is recognized by MMP-2. Again, we consid-

ered the two possible locations of the N-terminal frag-

ment. Along the simulations of the MMP-2/peptide com-

plexes, we characterized the structural and dynamical

changes on substrate binding, the specific role of the key

MMP-2 residues in anchoring the substrate, and the

influence of the N-terminal fragment on the stability of

the enzyme/substrate interactions. The relative binding

free energy of the two complexes was also estimated by

means of the MM-PBSA calculations. Altogether these

theoretical results provide insight into the catalytic pro-

cesses taking place in the active site of the MMP-2

enzyme and can also be useful for the structural-based

design of specific MMP inhibitors.

MATERIALS AND METHODS

Setup of the systems

Initial coordinates for the MMP-2 catalytic domain

were taken from the 1CK7 crystal structure, which corre-

sponds to the Ala404Glu mutant of the full-length pro-

MMP-2 enzyme.10 The propeptide (Pro31-Asn109) and

the C-terminal hemopexin-domain (Leu461-Cys660) were

removed from the initial structure, while the three fibro-

nectin-type domains (Glu217-Gln393) that characterize

gelatinases were replaced by a short peptide segment (Lys-

Gly-Val) using the 1QIB crystal structure as a template.12

Two zinc and two calcium ions were bound to the cata-

lytic domain as observed in the initial 1CK7 structure and

in our previous computational study.15 The ionizable resi-

dues were set to their normal ionization states at pH 7.

To generate the ‘‘N-terminal disordered’’ and the

‘‘superactived’’ forms of the catalytic domain of MMP-2,

we built two protein models differing in the initial posi-

tioning of the N-terminal coil. The initial orientation of

the ‘‘disordered’’ N-terminal fragment corresponds to

that observed in the 1CK7 structure, in which the amino

acids Tyr110-Lys118 do not present significant contacts

with other residues of the catalytic domain.10 This

resulted in the initial N-UP configuration (see Fig. 1).

On the other hand, the 2CLT crystal structure of the

MMP-1 enzyme displays an N-terminal fragment that is

well ordered within the hydrophobic cleft defined by the

a3-helix and the X-loop, and forms one salt-bridge and

several H-bond contacts with other residues of the

enzyme.21 To generate an initial structure for the cata-

lytic domain of the MMP-2 enzyme with the N-terminal

fragment sandwiched between the a3-helix and the X-loop (the N-DOWN model), we superposed the 2CLT

structure onto the 1CK7 one by minimizing the Root

Mean Square deviation (RMSD) of the backbone atoms

of the residues that coordinate the two zinc ions (RMSD

0.45 A). Subsequently, the N-DOWN model of the

MMP-2 enzyme was derived by replacing the coordinates

of the backbone atoms of residues Tyr110-Lys118 with the

corresponding ones in the 2CLT structure (Phe81-Arg89).

Side chain atoms for the Tyr110-Lys118 residues in the

N-DOWN model were constructed using the LEaP pro-

gram.22 Because the LEaP program does not select

between side chain rotamers to avoid steric clashes, the

system was properly relaxed by energy minimizations

before starting the simulations. Two water molecules

trapped within a cavity formed around the N-terminal

ammonium group in the 2CLT structure21 were also

included in the N-DOWN model.

Docking Calculations

To obtain an initial structure for the complex formed

between the catalytic domain of the MMP-2 enzyme and

the Ace-Gly-Pro-Gln-Gly�Ile-Ala-Gly-Gln-Nme peptide

substrate (the C1 peptide), we followed a ‘‘mixed’’

approach in which the valuable structural information

provided by the crystal structures of several MMP com-

plexes with different types of peptide-like inhibitors1,14

was merged with the results of automatic docking calcu-

lations. First, the backbone of the peptide substrate was

manually built in an extended b-conformation and

placed within the MMP-2 active site of the previously

edited 1CK7 X-ray structure (i.e., in the N-UP model).

The conformation of the non-primed moiety of C1 (P4–

P1) was then refined using the 1JAP crystal structure as a

template, which corresponds to the complex formed

between the MMP-8 enzyme and the nonprimed (P3–P1)

Pro-Leu-Gly-hydroxamic acid inhibitor.23 In this way,

the C1 proline residue in P3 was properly placed within

the S3 hydrophobic pocket defined by the Tyr182, His193,

and Phe195 side chains, and the P1 carbonyl group was

positioned close to the catalytic zinc ion (Zn1). The Gly-

Pro (P4–P3) peptide bond of C1 was manually built in

the most frequent trans conformation. For the primed

moiety of the substrate (P 01–P

04), our initial model estab-

lished H-bond contacts with several backbone groups

N. Dıaz and D. Suarez

52 PROTEINS

from the b4-strand and the X-loop of MMP-2, in agree-

ment with the available crystal structures for primed-

inhibitors.14 In addition, the P01 side chain of C1 was

located inside the S01 hydrophobic channel.To further refine the contacts between the peptide sub-

strate and the enzyme, as well as to perform a conforma-

tional sampling of the two ends of the C1 peptide (Ace-

Gly2 and 2Gln-Nme) for which no previous structural

information was available, we employed the LMOD pro-

gram linked to the AMBER package.22 LMOD imple-

ments a conformational search algorithm based on eigen-

vector following of low frequency vibrational modes that

allows flexible docking and protein loop optimization.24

During the LMOD calculations, all the protein residues,

except the N-terminal coil and the residues coordinating

the zinc and calcium ions, were allowed to move. We

employed the parm94 force field and a distant-dependent

dielectric constant to mimic solvent effects. A total of

2400 LMOD iterations were computed by exploring 12

low-frequency vibrational modes. Eigenvectors were

recalculated every 10 LMOD iterations. In addition to

the internal motions, we applied four rigid body rota-

tional and translational motions to the ligand at each

LMOD iteration.

The LMOD calculations generated a total of 50 low

energy structures for the MMP-2/C1 complex. Inspection

of these structures confirmed that the C1 substrate main-

tains the main enzyme/ligand contacts that were present

in the initial structure. The structure with the lowest

LMOD energy was then selected to build the N-UP/C1

and N-DOWN/C1 models following the same prescrip-

tions that were used for the native form of the enzyme.

MD Simulations

The initial structures of the N-UP, N-DOWN, N-UP/

C1, and N-DOWN/C1 models were surrounded by a per-

iodic box of TIP3P water molecules that extended 15 A

from the protein atoms. In addition, counterions were

placed to neutralize the systems. This resulted in a total

of 2527 protein atoms being solvated by �11,500 water

molecules. The parm94 version of the all-atom AMBER

force field was used to model the system.25 For the cal-

cium ions, we employed the nonbonded representation

proposed by Aqvist.26 For the zinc ions, we used a set of

MM parameters that have been developed and tested by

us in a previous work.15 Particularly for the catalytic

Zn1, a mixed bonded and nonbonded representation is

adopted in which the metal ion is linked to the His403-

Ne, His407-Ne, His413-Ne atoms and the bridging

[Zn��(OH2)]21���2OOC��Glu404 water molecule by

explicit MM bonds. In contrast, the fifth ligand, a TIP3P

water molecule in the N-UP and N-DOWN models or

the P1 carbonyl group of the C1 peptide in N-UP/C1

and N-DOWN/C1, is represented by the standard non-

bonded parameters. This mixed description allows sol-

vent exchange and flexible substrate binding at the apical

position of the Zn1 site. For the tetrahedral Zn2 site, we

employed a fully bonded representation in which Zn2was bonded to the His178-Ne, Asp180-Od, His193-Ne, andHis206-Nd atoms. This Zn2 parameterization resulted in a

stable tetrahedral coordination environment as observed

in the 1CK7 crystal structure. Further details of the Zn1/

Zn2 parameterization (reference bond lengths, atomic

charges, etc.) can be found elsewhere.15

In our previous work on the MMP-2 enzyme,15 the

force field parametrization of Zn1 and Zn2 in the free form

of the MMP-2 enzyme has been tested by performing

energy minimizations and short MD simulations, showing

that the average Zn-ligand distances and angles extracted

from the MD simulations remain close to the reference

QM/MM values. In this work, the quality of the Zn1 repre-

sentation in the presence of the C1 peptide was similarly

assessed by performing a QM/MM energy minimization of

the Michaelis complex in the N-UP/C1 model. The com-

parison of the Zn-ligand distances and angles computed

with the hybrid DFT/MM method and the average values

obtained from a short MD simulation of the complex fur-

ther validates the force field representation employed for

Zn1 (see Supporting Information for details).

Energy minimizations and MD simulations were car-

ried out using the SANDER and PMEMD programs

included in the AMBER 9.0 suite of programs.22 The

solvent molecules and counterions were initially relaxed

by means of energy minimizations and 50 ps of MD.

Subsequently, the full systems were minimized to remove

bad contacts in the initial geometry and heated gradually

to 300 K during 60 ps of MD. The SHAKE algorithm

was employed to constraint all R–H bonds, and periodic

boundary conditions were applied to simulate a continu-

ous system. A nonbonded cutoff of 10.0 A was used

whereas the Particle-Mesh-Ewald (PME) was employed

to include the contributions of long-range interactions.27

The pressure (1 atm) and the temperature (300 K) of the

system were controlled during the MD simulations by

Berendsen’s method.28 A 20 ns trajectory was computed

for each model with a time step of 2 fs. Coordinates

were saved for analysis every 1 ps.

Only the last 15.0 ns of each trajectory were analyzed

using the CARNAL module of AMBER and some other

specific software developed locally. The solvent accessible

surface area (SASA) of the MMP-2 systems was com-

puted using the MSMS program.29 We also computed

the radius of accessibility to important anchorage points

along the active site cleft as described previously.15

Structural figures were produced with the programs Mol-

script and Raster3D.30,31

Energetic analyses of the MD trajectories

The MM-PBSA approach can perform several types of

free energy calculations (protein–protein binding ener-

N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 53

gies, conformational energies of protein loops, etc.).32,33

Basically, the MM-PBSA calculations predict mean values

of free energies as estimated over a series of representa-

tive snapshots extracted from classical MD simulations.

The snapshots are postprocessed through the removal of

all solvent and counterions. Then one calculates the aver-

age free energy of the set of structures according to the

following equation:

G � EMM þ 3RT þ GPBSA � TSMM ð1Þ

where G is the estimated average free energy and EMM is

the average molecular mechanics energy,

EMM ¼ Ebond þ Eangle þ Etors þ EvdW þ Eelec ð2Þ

where these correspond to the bond, angle, torsion, van

der Waals, and electrostatic terms in the molecular

mechanics force field. The term 3RT in Eq. (1) is the en-

thalpy of the six translation and rotational degrees of

freedom in the classical limit. GPBSA is the solvation free

energy obtained from Poisson-Boltzmann electrostatic

calculations augmented with an estimate of the nonpolar

free energy via molecular area, and 2TSMM is the solute

entropy which can be estimated by molecular mechanics

normal mode calculations and standard statistical me-

chanical formulae.32 Subsequently, one can estimate the

DG for ligand association to proteins using the following

equation:

DG ¼ Gcomplex � Gprotein � Gligand ð3Þ

where the three G terms are usually evaluated using the

snapshots from a single MD trajectory of the complex

(the one trajectory approximation). Here, the binding

free energies are computed for a standard state of 1 M.

As a consequence, the translational entropy for each

component (complex, protein, ligand) is 6.4 cal/(mol K)

smaller than the entropy value obtained for the standard

state of an ideal gas owing to the change in concentra-

tion from 0.045 M (ideal gas) to 1 M (solution).33

In this work, a set of 300 representative structures

extracted every 50 ps along the MD trajectories were

postprocessed to calculate the free energies of the cata-

lytic domain of the MMP-2 enzyme. The AMBER force

field was used to compute (no cutoff) the EMM terms

defined in Eq. (2). The electrostatic contributions to the

solvation free energy were determined with the Poisson-

Boltzmann approach, which represents the solute as a

low dielectric continuum (a value of eint 5 1 was used in

the calculations) with embedded charges and the solvent

as a high dielectric continuum (eout 5 80) with no salt.

Atomic charges and radii were taken from the AMBER

representation of the MMP-2 models. The dielectric

boundary is the contact surface between the radii of the

solute and the radius (1.4 A) of a water probe molecule.

The PBSA program included in the AMBER 9.0 pack-

age22 was employed to solve the linearized PB equation

on a cubic lattice with a grid spacing of 0.5 A.

Solute entropic contributions were estimated using the

NMODE module of the AMBER 9.0 package.22 The

nmode program uses the normal modes and standard

statistical thermodynamic formulae to estimate entropic

contributions (due to the high computational demand,

normal mode calculations were performed only for 100

snapshots). Before the normal mode calculations, the

geometries of the MMP-2 models described by their

AMBER representations were minimized until the root-

mean-square deviation of the elements in the gradient

vector was less than 1025 kcal/(mol A). The ROAR 2.0

program34 was used to carry out the geometry optimiza-

tions driven by a limited memory BFGS minimizer. All

minimizations and normal mode calculations were car-

ried out with a distance-dependent dielectric constant (e5 4r) to mimic solvent screening with no cutoff for the

nonbonded interactions. As noted in previous work,35

this normal mode analysis only approximately estimates

solute entropy.

Validation Calculations

The relative stability of the N-UP and N-DOWN

MMP-2 models is mainly determined by their relative

solvation energies and the strength of intraprotein and

enzyme–substrate contacts. All these interactions can be

taken into account by means of the MM-PBSA calcula-

tions. However, to better calibrate the performance of the

MM-PBSA approach for our particular problem, we car-

ried out a series of test calculations using both quantum

chemical and MM-PBSA methodologies to estimate the

average interaction energy between the first 10 amino

acid residues of the MMP-2 enzyme (i.e., the N-terminal

fragment) and a protein subsystem formed by residues of

the a3-helix and the X-loop that interact with the N-ter-

minal fragment along the N-DOWN simulation (His407,

Ala408, Met409, Gly410, Leu411, Glu412, His413, Ser414,

Asp436, Asp437, Lys439, Gly440, Glu443, Leu444). Terminal

N-methylamine or acetyl groups were placed at the C-

and N-backbone atoms of those residues cleaved from

the protein main chain by the truncation process (see

Fig. S2 in the Supporting Information).

The DFT energies were obtained using the TURBO-

MOLE suite of programs.36 We employed the PBE func-

tional37 combined with a double-f plus polarization

basis set (SVP).38 The DFT interaction energies in the

gas-phase were corrected for the Basis Set Superposition

Error by means of the counterpoise method. To estimate

the effect of the solvent environment on the DFT

energies, we used the conductor-like screening model

(COSMO) included in TURBOMOLE in which the sol-

vent dielectric continuum is approximated by a scaled

conductor.39 All the DFT calculations were performed in

N. Dıaz and D. Suarez

54 PROTEINS

the framework of the multipole accelerated ‘‘resolution-

of-the-identity’’ approximation (MARI-J) using the

appropriate auxiliary basis set.40,41

Since the PBE gradient-corrected density functional is

unable to describe dispersive interactions, the DFT energy

terms were augmented with an dispersion energy contri-

bution, Edisp, that was computed using an empirical for-

mula that has been introduced by Elstner et al.42 to

extend their approximate DFT method43 for the descrip-

tion of dispersive interactions. The Edisp expression con-

sists basically of a C6/R6 term, which is appropriately

damped for short R distances. We used the same parame-

ters for C, N, O, S, and H and combination rules as

those described by Elstner et al.42

A total of 25 interaction energy calculations were per-

formed on coordinates extracted from the N-DOWN

MD trajectory at time intervals of 50 ps. For each one of

the 25 structures, the interaction energy (DEint) between

the N-terminal fragment and the selected protein residues

in a3-helix and the X-loop was computed by means of

the MM-PBSA and DFT-based methodologies (see Table S2

in the Supporting Information). The MM-PBSA and DFT

DEint data are correlated to each other, the corresponding

linear correlation coefficient being 0.933. The resultant av-

erage values for the interaction energy (DEint) were 243.4

and 247.7 kcal/mol at the MM-PBSA and the DFT levels,

respectively, the standard error of the DEint values being 1.0

and 1.7 kcal/mol. Given that the unsigned difference

between the MM-PBSA and DFT values (4.3 kcal/mol) is

not large, we expect that the MM-PBSA method can pre-

dict relative interaction energies comparable with those pre-

dicted by the more sophisticated PBE method.

RESULTS AND DISCUSSION

MD simulations of the native form of MMP-2

In Table I, we collect the Root Mean Squared Deviation

(RMSD) and the Root Mean Squared Fluctuation

(RMSF) values obtained for the N-UP and N-DOWN tra-

jectories of the native enzyme. We found that the main

differences in the backbone RMSDs between the two

models arise in the N-terminal coil (2.61 � 0.31 A versus

3.30 � 0.10 A), in the b3-strand (0.88 � 0.31 A vs. 0.46

� 0.12 A), and in the b4–b5 connecting loop (0.66 �0.10 A vs. 1.02 � 0.31 A). With respect to the local flexi-

bility, the N-terminal coil becomes clearly more ordered

along the N-DOWN trajectory, in which it remains well

positioned within the hydrophobic crevice defined by the

a3-helix and the X-loop (see Fig. 1). Thus, the backbone

RMSF values of the N-terminal coil shift from 1.09 �0.37 A for the N-UP simulation to 0.41 � 0.09 A for the

N-DOWN model. In turn, the b4–b5 connecting loop

becomes significantly more flexible along the N-DOWN

simulation (from the 0.29 � 0.07 A measured for N-UP

to 0.60 � 0.17 A along N-DOWN). The radius of gyra-

tion and the Solvent Accessible Surface Area (SASA) val-

ues confirm that the N-DOWN model is more compact

than the N-UP one. The main differences in the local

contributions to the SASA values arise in the a3-helix, inthe N-terminal coil, and in the X-loop (see Fig. 1)

because these secondary structure elements become less

solvent exposed on going from N-UP to the N-DOWN

model. In contrast, the b4–b5 connecting loop is more

solvent accessible along the N-DOWN trajectory.

In Figure S3 of the Supplementary Material, we repre-

sent the superposition of the average structures obtained

from the N-UP and N-DOWN simulations onto the ini-

tial X-ray structure. Inspection of this figure shows that,

along the two simulations, only the first part of the N-

terminal coil (nine residues: from Tyr110 to Lys118)

diverges significantly from the initial X-ray structure. The

stability of the Trp119��NH���Oh��Tyr445�H��bond con-

tact (97%, 3.07 � 0.16 A for N-UP and 94%, 3.13 �0.16 A for N-DOWN) determines that both average

structures clearly superpose onto the X-ray one at this

position. For the N-UP model, the first nine residues of

the N-terminal coil give only two contacts with the rest

of the catalytic domain: the Lys116 backbone contacts the

Pro197 carbonyl group from the b4-b5 connecting loop

(2.97 � 0.15 A and 99% of occupancy), while Phe113interacts with the Phe195 aromatic ring that borders the

Table IAverage Values and Standard Deviations for RMSD, RMSF, Radius of Gyration,

and Solvent Accessible Surface Area (SASA) Derived from the MD Simulations

N-UP N-DOWN N-UP/C1 N-DOWN/C1

RMSD (�)All heavya 2.01 � 0.16 2.02 � 0.10 1.96 � 0.11 2.04 � 0.09Backbonea 1.52 � 0.14 1.52 � 0.11 1.42 � 0.08 1.55 � 0.09Helix a2b 0.37 � 0.04 0.40 � 0.05 0.41 � 0.05 0.38 � 0.05Helix a3b 0.73 � 0.07 0.71 � 0.06 0.75 � 0.07 0.71 � 0.06Strand b4b 0.23 � 0.06 0.25 � 0.06 0.23 � 0.05 0.22 � 0.04N-term coil 2.61 � 0.31 3.30 � 0.10 2.46 � 0.28 3.41 � 0.12Zn/Ca S loopb 1.28 � 0.12 1.29 � 0.19 1.55 � 0.15 1.23 � 0.11b4-b5 loopb 0.66 � 0.10 1.02 � 0.31 0.53 � 0.11 1.04 � 0.48X loopb 1.31 � 0.20 1.20 � 0.19 0.98 � 0.14 1.19 � 0.14

RMSF (�)All heavya 1.23 � 0.14 1.01 � 0.11 0.98 � 0.09 1.01 � 0.12Backbonea 0.94 � 0.15 0.81 � 0.09 0.68 � 0.08 0.73 � 0.08Helix a2b 0.24 � 0.04 0.24 � 0.05 0.24 � 0.04 0.24 � 0.05Helix a3b 0.35 � 0.08 0.30 � 0.08 0.31 � 0.08 0.27 � 0.06Strand b4b 0.18 � 0.07 0.18 � 0.06 0.15 � 0.05 0.13 � 0.04N-terminal coilb 1.09 � 0.37 0.41 � 0.09 1.10 � 0.20 0.43 � 0.11Zn/Ca S loopb 0.73 � 0.23 0.69 � 0.14 0.55 � 0.12 0.42 � 0.10b4-b5 loopb 0.29 � 0.07 0.60 � 0.17 0.32 � 0.08 0.73 � 0.18X loopb 0.67 � 0.18 0.64 � 0.18 0.61 � 0.14 0.65 � 0.19

Radius of gyrationc (�)15.3 � 0.1 15.2 � 0.0 15.5 � 0.2 15.1 � 0.0

SASA (�2)9376 � 163 9074 � 133 9517 � 146 8974 � 144

aWithout including the random N-terminal coil (residues Asn111-Asn122).bCorresponding to the backbone heavy atoms.cX-ray value (1CK7) 14.8 A.

N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 55

S3 hydrophobic pocket (5.23 � 0.95 A between the centre

of mass of the phenyl rings). In contrast, the N-terminal

fragment establishes very stable H-bond contacts with

several residues from the a3-helix and the X-loop during

the N-DOWN simulation. Particularly, the Tyr110��NH3

1���2OOC��Asp436 salt bridge between the N-

terminal ammonium group and the conserved aspartic

acid placed in the a3-helix is well maintained during the

N-DOWN simulation (2.83 � 0.13 A and 100% of occu-

pancy). In addition, Asn111 contacts with Glu412 from the

X-loop through a direct Asn111��O���HN��Glu412 H-

bond interaction (2.94 � 0.14 A and 100% of occu-

pancy) and through a buried water molecule (Asn111��NH���Wat���O��Glu412, 99% of occupancy). Similarly, the

Phe113 amino group remains H-bonded to the Gly410 car-

bonyl group placed at the beginning of the X-loop (2.99

� 0.15 A and 99% of occupancy) all along the N-

DOWN trajectory.

In Table II, we show the radii of accessibility15 com-

puted for important substrate anchorage points located

in the MMP-2 active site cleft. The values in Table II

confirm that the binding sites for the peptide backbone

groups (see Scheme 1) are readily accessible along the N-

UP and N-DOWN trajectories. For the S3 hydrophobic

pocket bordered by the Tyr182, His193, and Phe195 rings,

however, the radius of accessibility computed for the

His193 side chain increases significantly ongoing from N-

UP (3.28 � 1.10 A) to N-DOWN (4.67 � 0.66 A), sug-

gesting that the position adopted by the N-terminal coil

can affect the dimension and stability of this site. In fact,

we see in Figure 2 that the relative orientation of the

Tyr182, His193, and Phe195 side chains depends on the

positioning of the N-terminal coil. Thus, two hydropho-

bic contacts are observed in the N-UP simulation: (a) the

Phe113 side chain (N-terminal coil) pairs with Phe195(S3); (b) the Tyr182 phenol group partially overlaps the

His193 side chain (4.65 � 0.98 A). In the N-DOWN con-

Table IIAverage Values and Standard Deviations for the Radii of Accessibility (A) of

Important Anchorage Points Located in the Binding Site Cleft of the MMP-2

Catalytic Domain

N-UP N-DOWN N-UP/C1a N-DOWN/C1a

Gly189 CO 4.72 � 1.00 5.78 � 0.43 3.81 � 0.55 4.14 � 0.74Leu191 N 2.44 � 0.37 2.60 � 0.45 2.22 � 0.19 2.34 � 0.15Ala192 N 2.13 � 0.30 1.83 � 0.31 2.24 � 0.14 2.23 � 0.18Ala192 CO 2.51 � 0.40 3.84 � 0.33 2.70 � 0.34 2.43 � 0.18His193 3.28 � 1.10 4.67 � 0.66 5.11 � 0.47 5.24 � 0.37Ala194 N 2.15 � 0.31 2.20 � 0.32 2.16 � 0.26 1.90 � 0.19Ala194 CO 4.96 � 0.62 4.03 � 0.70 5.62 � 0.41 4.53 � 0.45His403 2.53 � 0.34 2.74 � 0.27 2.28 � 0.13 2.34 � 0.14His407 CO 2.21 � 0.34 1.70 � 0.18 1.97 � 0.33 1.74 � 0.37Gly410 CO 3.77 � 0.70 1.22 � 0.16 2.71 � 0.46 0.95 � 0.14Ala419 CO 1.14 � 0.25 1.56 � 0.37 2.30 � 0.43 1.66 � 0.29Pro423 CO 5.03 � 1.10 5.71 � 0.52 3.30 � 0.53 4.17 � 0.91Tyr425 N 3.27 � 0.59 4.49 � 1.01 2.74 � 0.32 2.56 � 0.26Val400 (S01)

b 2.36 � 0.29 2.27 � 0.21 2.17 � 0.13 2.30 � 0.12Val400 (X)

c 0.76 � 0.22 0.78 � 0.19 1.67 � 0.33 1.11 � 0.22

aThe radii of accessibility of N-UP/C1 and N-DOWN/C1 were computed after

deleting the coordinates of the C1 peptide.bWith the X-loop secondary door blocked.cWith the S01 main-door blocked.

Figure 2Structure of the S2 region and the S3 hydrophobic pocket as observed in representative snapshots from the N-UP and N-DOWN simulations.

N. Dıaz and D. Suarez

56 PROTEINS

figuration, the Tyr182, His193, and Phe195 side chainsadopt a more solvent accessible conformation (see Fig. 2)while the Phe113 side chain extends alongside the S2region, approximately bordered by residues from His407to His413,44 thus reducing the accessibility of some S2subsites (see the changes in the radii of accessibility ofthe His407��CO, and Gly410��CO groups in Table II).

In the crystal structure of the MMP-2 enzyme, the S01pocket is a channel that connects a main entrance located

adjacent to the catalytic Zn1 ion with a secondary door

encircled by residues from the X loop.15 We found that the

accessibility of the S01 pocket is hardly influenced by the

positioning of the N-terminal coil as shown by the racc val-

ues of the Val400 and His403 side chains (see Table II). Thus,

the His403 side chain, which is placed at the S01 main en-

trance, displays similar racc values along both the N-UP

(2.53 � 0.34 A) and N-DOWN (2.74 � 0.27 A) simulations.

Similarly, Val400, located inside the S01 site, is also readily ac-

cessible from the active site cleft (2.36 � 0.29 A for N-UP

and 2.27 � 0.21 A for N-DOWN). However, we note that

the racc value of Val400 passing though the secondary door is

reduced to 0.8 � 0.2 A, that is, the S01 site becomes a closed

pocket during the N-UP and N-DOWN simulations.

MM-PBSA calculations of N-UPand N-DOWN

As the backbone conformation of the N-terminal resi-dues remained quite stable along the last 15 ns of the N-UP and N-DOWN MD simulations, it is likely that theN-terminal coil is trapped in two different local freeenergy minima. To estimate the relative stability of thetwo conformers of the N-terminal coil, we computed theMM PBSA free energy of the N-UP and N-DOWN mod-els. For systems of similar size, it has been shown thatthe correlation time for decay of fluctuations of the sumof the EMM and GPBSA energies is about 1 ps.33 Thus, weexpect that the N-UP and N-DOWN snapshots used forfree energy evaluation, which were extracted at timeintervals of 50 ps, are independent.

Table III contains the average values of the energetic

terms that are combined to estimate the MM-PBSA free

energy for the N-UP and N-DOWN MMP-2 configura-

tions. From the Gtotal values in Table III, the relative sta-

bility of the two models can be assessed directly. Thus, it

turns out that the interaction of the N-terminal coil with

the a3-helix and the X-loop (N-DOWN) is energetically

more favourable than the more solvent-accessible orienta-

tion (N-UP) by 13 kcal/mol. This energy difference is sig-

nificantly larger than the standard errors and, therefore,

N-DOWN would be the most likely model representing

the native form of the MMP-2 catalytic domain. Accord-

ing to the relative values of the free energy components

in Table III, the larger stability of the N-DOWN model

stems from stronger intraprotein contacts accounted for

by the Eelec and EvdW terms, which overcompensate both

the entropic and desolvation penalties associated to the

N-UP?N-DOWN process. This is in consonance with

the structural analyses that characterize N-DOWN as the

most compact and less flexible configuration.

MD simulations of the MMP-2/peptidecomplexes

Binding of the Ace-Gly-Pro-Gln-Gly�Ile-Ala-Gly-Gln-

Nme peptide (labeled as C1) to the MMP-2 active site

does not induce significant changes in the global struc-

ture of the host enzyme. In Table I, we see that the

RMSD values obtained for the N-UP/C1 and N-DOWN/

C1 trajectories are similar to those obtained for the N-UP

and N-DOWN simulations of the native enzyme, res-

pectively. Concerning protein mobility, the presence of

the C1 peptide slightly reduces the flexibility of the

N-UP configuration (from 0.94 � 0.15 A to 0.68 � 0.08 A)

whereas the global RMSF values are more comparable for

the two N-DOWN models (0.81 � 0.09 A and 0.73 �0.08 A).

Figure 3 shows the positioning of C1 in the MMP-2active site and the main H-bond contacts that contribute

Table IIIAverage Values for the MM-PBSA Free Energy Components (kcal/mol) of the MMP-2 Models

EMM Eelec EvdW DGPBSA 2TSMM Gtotal

N-UP 22780.4 (5.4) 24935.6 (4.9) 2637.9 (1.2) 22228.6 (4.2) 21837.8 (0.7) 26845.0 (3.0)0.0

N-DOWN 22940.6 (4.1) 25073.4 (3.7) 2655.8 (1.1) 22090.7 (3.3) 21828.5 (0.5) 26858.2 (2.6)2160 2138 218 138 9 213

N-UP/C1 22982.9 (4.5) 25201.0 (4.2) 2698.9 (1.1) 22192.6 (4.2) 21919.3 (0.7) 27093.0 (2.9)0.0

N-DOWN/C1 23196.4 (4.4) 25391.2 (3.5) 2729.9 (1.2) 22021.9 (3.4) 21908.8 (0.5) 27125.3 (3.0)2213 2190 231 171 10 232

DEMM DEelec DEvdW DGPBSA 2TDSMM DGbinding

N-UP/C1 2158.9 (0.8) 289.2 (0.8) 269.7 (0.2) 108.2 (0.7) 36.3 (0.6) 214.3 (0.9)N-DOWN/C1 2166.8 (0.6) 296.8 (0.6) 269.9 (0.2) 104.9 (0.6) 32.8 (0.4) 229.0 (0.7)

Relative differences are in italics. Standard errors of average values are indicated in parentheses.

N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 57

to the binding of the peptide substrate along the N-UP/C1 and N-DOWN/C1 trajectories. From the docking cal-culations followed by the 20 ns MD simulations, we pre-dict that C1 binds to MMP-2 in an extended conforma-tion that is stabilized by the following H-bond contacts(67–100% of occupancy) connecting the important back-bone positions in the b4-strand and the X-loop of theenzyme with the corresponding backbone amide groupsof C1: Ala194��O���HN��Gln(P2), Ala194��NH���O��

Gln(P2), Ala192��O���HN��Ile(P01), Leu191��NH���O��Ile(P01), Pro423��O���HN��Ala(P02), Tyr425��NH���O��Ala(P02), Gly189��O���HN��Gly(P03). These interactions areequally stable regardless of the UP/DOWN conformationof the N-terminal coil as the relatively short C1 peptideand the N-terminal coil do not give any direct contact.However, it is clear that binding of larger and more com-plex substrates like collagen could be sterically moreimpeded in the N-UP model (see Fig. 3).

Figure 3Ribbon models and molecular surface representations of the last snapshot from the N-UP/C1 and N-DOWN/C1 trajectories (the C1 peptide and the N-terminal coil are

depicted in sticks with carbon atoms shown in green and orange, respectively). Schematic representation of the main enzyme/substrate binding determinants (average

distances are in A).

N. Dıaz and D. Suarez

58 PROTEINS

The most important difference in the MMP-2/C1 polar

interactions between the N-UP/C1 and N-DOWN/C1 sim-

ulations corresponds to the contacts established by the P2glutamine side chain, which points towards the broadly

defined S2 binding site. Thus, the Gln(P2) side chain, that

can rotate freely along the N-UP/C1 trajectory, gives only

a weak His407��NdH���Oe��Gln(P2) interaction (42%,

3.10 � 0.21 A). However, in the N-DOWN/C1 simulation,

the Gln(P2) side chain is basically locked in a stable

Gln(P2)��NeH��e��Glu412 H-bond contact (86%, 2.97 �0.21 A). The origin of this difference in the enzyme/sub-

strate contacts at P2 can be traced back to the positioning

adopted by the N-terminal coil. In the N-UP/C1 configu-

ration, the Glu412 side chain is fully solvent accessible and

scarcely interacts with other residues. In contrast, a short

segment of the N-terminal coil (Asn111-Phe113) is located

around the Glu412 side chain along N-DOWN/C1 (see Fig.

3), restricting thus the mobility of this residue and favour-

ing its interaction with the Gln(P2) side chain.

Hydrophobic contacts contribute also to anchor the C1

substrate within the MMP-2 active site. Thus, the P3 proline

residue remains accommodated in the S3 hydrophobic

pocket during the two simulations of the MMP-2/C1 com-

plex. The distances between the center of mass of the side

chains bordering the S3 pocket (Tyr182, His193, and Phe195)

and the P3 proline ring vary between 4.3 and 6.1 A for the

N-UP/C1 simulation, and 4.2-5.1 A for the N-DOWN/C1

trajectory. In addition, the P01 leucine side chain is located

in the S01 hydrophobic pocket interacting with the Leu191,

Ala192, and Val400 side chains (the distances between the

centre of mass of the side chains lie in the 5.1–5.6 A range).

To find out to what extent the presence of the C1 sub-

strate influences the accessibility and shape of the enzyme

binding sites, it can be useful to compare the corre-

sponding radii of accessibility computed for both the

unbound and the C1-bound MMP-2 models (the coordi-

nates of C1 were removed before the racc calculations, see

Table II). For the backbone anchorage points located in

the b4-strand and in the X-loop (Leu191, Ala192, Ala194,

Pro140, etc.), their racc values in the N-UP/C1 and N-

DOWN/C1 configurations tend to have lower average

values (�2.2 A) and to exhibit lower fluctuations than in

the unbound models. Notwithstanding, the accessibility

of the majority of these sites is very similar in all the

simulations, showing thus that the unbound enzyme is

well preorganized to give these important H-bond con-

tacts. Perhaps the observed changes in the shape of the

S03 and S1 hydrophobic pockets can be of more interest.

Thus, the racc values suggest that the S03 pocket becomes

larger when it is occupied by the proline P3 residue, what

was confirmed by visual inspection. We also note that

the architecture of the occupied S3 pocket does not

depend on the location of the N-terminal coil, in con-

trast with the results obtained for the unbound models.

Concerning the S01 pocket, it turns out that the presence

of the isoleucine P01 side chain favours the opening of its

secondary entrance as shown by the computed racc values

for Val400 when the main door is blocked, which amount

to 1.67�0.33 A (N-UP/C1) and 1.11�0.22 A (N-DOWN/

C1) (see Fig. 3). The observed flexibility of the S01 and S3sites on substrate binding suggest that protein/ligand

docking analyses, which normally consider the protein to

be rigid, should be treated with caution when analyzing

the ligand interactions with these hydrophobic pockets.

MM-PBSA calculations of N-UP/C1and N-DOWN/C1

As shown in Figure 3, the positioning of the N-termi-

nal coil results in a moderate effect on the nature and

structure of the C1 binding determinants, the main dif-

ference arising at the level of the Gln(P2) side chain con-

tacts. However, the MM-PBSA energetic analyses sum-

marized in Table III point out that, on substrate binding,

the packaged conformation of the N-terminal coil

becomes more stable by about 19 kcal/mol with respect

to the unbound form of MMP-2. When we analyze the

corresponding changes in the different free energy com-

ponents, we see that the presence of C1 modifies the rel-

ative values of Eelec, EvdW, and GPBSA by 252, 213, and

133 kcal/mol, respectively, showing thus that the electro-

static intra-protein contacts are most reinforced in the

N-DOWN/C1 model. Reciprocally, the MM-PBSA bind-

ing free energy between the MMP-2 catalytic domain and

the C1 peptide is about 15 kcal/mol larger in absolute

value in the N-DOWN/C1 model than in N-UP/C1. In

this case, the electrostatic and desolvation contributions

tend to favor the binding of the C1 peptide when the

N-terminal coil is in its ‘‘superactivated’’ form. Part of the

energetic stabilization of the N-DOWN/C1 model can be

due to the Gln(P2)��NeH��e��Glu412 interaction that is

formed only in this configuration. However, besides this

local interaction, it is likely that the N-terminal coil posi-

tioning and the C1 binding can influence each other

through medium-range electrostatic interactions, which

should stabilize preferentially their approximate antiparallel

alignment as in the N-DOWN/C1 configuration (see Fig. 3).

At this point, it may be interesting to note that the

predicted energetic preference for positioning the N-ter-

minal coil within the hydrophobic groove defined by the

a3-helix and the X-loop, either in the unbound or in the

complexed form of MMP-2, seems in consonance with

the ‘‘superactivation’’ phenomenon observed for other

MMPs.16 Nevertheless, we also note that a full descrip-

tion of ‘‘superactivation’’ is beyond the scope of this

work because it involves kinetic effects and the considera-

tion of N-terminal coils of variable length.

Implications for the catalytic mechanism

All the described polar and hydrophobic contacts

between MMP-2 and C1 are compatible with the car-

N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 59

bonyl group of the Gly(P1) residue acting as the fifth

ligand around the catalytic Zn1 ion. Thus, the computed

average Gly(P1)��O���Zn1 distances are 2.25 � 0.14 A for

N-UP/C1 and 2.31 � 0.16 A for N-DOWN/C1 (note

that the Gly(P1)��O���Zn1 bond was not explicitly

defined in the force field representation of Zn1). In prin-

ciple, the polarization of the Gly(P1) carbonyl group

induced by the metal ion could facilitate the nucleophilic

attack by the Zn1-bound water molecule (Wat1), which

in turn, connects the Zn1 ion and the conserved Glu404side chain through a Zn1���OH2���Oe��Glu404 bridge.

Most interestingly, in our MD simulations, the average

Gly(P1)��C���O��Wat1 distance is only 2.97 � 0.11 A for

N-UP/C1 and 2.87 � 0.10 A for N-DOWN/C1, the cor-

responding values for the Gly(P1)��O���Gly(P1)��C���Wat1-O angle being (71 � 5)8 and (76 � 6)8. There-fore, it is clear that the C1 decapeptide adopts a reactive

configuration in the MMP-2 active site in which Wat1 is

properly oriented to readily attack the carbonyl group of

the peptide bond with the assistance of the conserved

glutamate residue (Glu404 in MMP-2), in agreement with

a previously proposed reaction mechanism for the MMP

enzymes.45 However, other reactive events along the cata-

lytic mechanism, like the protonation and/or departure of

the leaving amino group, may require some rearrangement

of the substrate within the active site groove and they

could determine the global rate of the catalytic process.

Implications for inhibitor design

On the basis of the structural and energetic results pre-

sented in this work, we propose that the N-DOWN/C1

configuration would be the most likely model for repre-

senting the reactive state of the MMP-2 catalytic domain.

This model not only complements the previous NMR

and X-ray structural information available for the MMP-

2 enzyme, either in its proactive form or in the presence

of inhibitors, but it can also be considered as a starting

point for the structure-based design of specific MMP-2

inhibitors.

On one hand, representative snapshots from the N-

DOWN/C1 MD simulation could be employed as tem-

plates in further computational drug discovery studies.

On the other hand, the MD results are useful to locate

more precisely the important binding determinants and/

or to assess the relative importance of the different con-

tacts. For example, the primed moiety of C1 (P01–P04)establishes five stable H-bond interactions with the

MMP-2 enzyme during the simulation, whereas the non-

primed residues (P1–P4) give only two or three (see Fig.

3). This computational observation is in agreement with

experimental results in which inhibitors that mimic the

sequence of the primed residues of peptide substrates ex-

hibit a better inhibition than those mimicking the ‘‘non-

primed’’ sites.1 However, it has also been observed that

sequence variability in the ‘‘nonprimed’’ sites is likely to

have an influence on substrate recognition across the

MMP family.1,44 Consequently, the ‘‘nonprimed’’ posi-

tions of peptidomimetic inhibitors could offer better

selectivity among the MMP enzymes and modulate the

primary binding force provided by Zn1-complexation

and occupation of the S01 pocket. For the MMP-2 enzyme,

kinetic experiments using a peptide phage library have

shown that P2 is a key residue in conferring selectivity

over the structurally related MMP-9 enzyme.46 Unfortu-

nately, the S2 site, which is located nearby the Zn1 ion,

comprises a relatively large and shallow region on the

MMP-2 surface bordered by seven residues (His407-

His413) in which well-defined anchorage points are not

easily distinguished a priori. Interestingly, the N-DOWN/

C1 simulation of the MMP-2 enzyme reveals that the

side chains of the P2 residue (Gln in C1) and the Glu412residue can form a stable interaction at the S2 site. This

interaction is promoted by the presence of part of the N-

terminal coil flanking the Glu412 side chain, altering thus

the structure of the S2 site with respect to the NMR and

X-ray structures. Our results suggest that optimization of

the P2���Glu412 interaction revealed by the N-DOWN/C1

simulation could lead to the design of novel inhibitors

with increased affinity for the MMP-2 enzyme.

ACKNOWLEDGMENTS

The authors thankfully acknowledge the computer

resources, technical expertise, and assistance provided by

the Barcelona Supercomputing Center – Centro Nacional

de Supercomputacion.

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N-Terminal Coil and Peptide Substrate in MMP-2

PROTEINS 61