matrix metalloproteinases 
 ( mmps ) are zinc - dependent endopeptidases 
 that play essential roles in various processes in the extracellular 
 matrix .
finding efficient ways to control 
 the mmp activity of gelatinases mmp-2 and mmp-9 , in particular , is 
 an important field of cancer research .
the inhibitory power of small organic compounds containing zinc - binding 
 groups ( carboxylate , thiolate , phosphinyl , hydroxamate ) is due to 
 the ability of these groups to chelate the catalytic zinc ion and 
 block the active site .
these compounds are characterized by a high 
 affinity for the zn ion but also often by a low specificity 
 for certain types of mmps , since all mmps possess similar active sites . to enhance the specificity of potential mmp inhibitors , oligopeptide - based 
 substances containing amino acid residues capable of interacting with 
 the zinc ion
together with the histidine residues from an 
 enzyme , these residues cooperate to perfectly match the typical coordination 
 shells of zn , whereas other residues interact with the 
 various binding sites of an enzyme that favor the specific affinities 
 of peptidomimetics .
one successful attempt in this direction was described 
 by hu et al . , who discovered regasepin1 , 
 a heptapeptide with inhibitory properties and a high selectivity to 
 mmp-9 .
however , they did not explain the origin of the inhibitory 
 power of regasepin1 , primarily because of a lack of structural data . 

aiming to clarify this issue , we considered a complex of mmp-9 with 
 regasepin1 by using advanced molecular modeling methods and discovered 
 that , upon binding of this peptide to the enzyme active site , a prominent 
 zinc finger motif pattern can be recognized .
this distinctive structural 
 motif of the cys2his2 class , with a tetrahedral 
 coordination of the zinc ion , is typical for multiple protein families 
 of cellular and transition factor types . in proteins ,
a zinc finger serves to stabilize the fold and to form 
 domains that bind specific parts of nucleic acids or other proteins . 
 unlike these constructs
, the zinc finger motif in the regasepin1mmp-9 
 complex is formed  on the fly  : an initial zn(his3 ) structural element in the enzyme active site is 
 rearranged to a more stable zn(cys2his2 ) pattern in the complex , in which two cys residues are borrowed 
 from the ligand .
motivated by this finding , 
 we deliberately constructed novel in 
 silico variants of peptidomimetics potentially capable of blocking 
 gelatinase a ( mmp-2 ) by forming the zinc finger motif via modifying 
 the zn(his3 ) pattern in the enzyme to zn(cys2his2 ) in the enzyme  ligand 
 complexes
. specifically , we designed and computationally characterized 
 a mutated variant of the known inhibitor app - ip , as well as a novel candidate constructed by mutating a 
 native substrate of mmp-2 .
one of the 
 problems in computational characterization of zinc - dependent 
 proteins by classical molecular mechanics ( mm ) or molecular dynamics 
 ( md ) methods is due to a large positive charge on the zn cation .
although fairly rigid structural fragments can be described 
 by classical force fields , the labile coordination shells surrounding 
 the catalytic zn ion in these enzymes must be described 
 via quantum methodology .
modern quantum chemical density functional 
 theory ( dft ) approaches provide valuable support to studies of metalloenzymes 
 ( e.g. , the recent review by blomberg , et al )
. structures of the zinc finger patterns and chemical reactions with 
 molecular clusters mimicking zinc finger constructs have been modeled 
 in only a few dft calculations .
development and practical implementation of multilevel 
 quantum mechanical / molecular mechanical ( qm / mm ) methods allows one to enhance simulations , in particular targeting structures 
 of zinc - containing proteins , and chemical 
 transformations in mmp active sites . including larger portions of 
 the protein provides better models to study binding patterns and transformation 
 of inhibitors that are not necessarily interacting directly with metal 
 ions and their immediate ligation sphere .
first , appropriate model 
 systems were constructed by motifs of the relevant crystal structures 
 from the protein data bank archive , and atomic coordinates were optimized 
 in qm / mm calculations .
parrinello 
 molecular dynamics with qm / mm potentials for model systems obtained 
 at the first stage were analyzed . for our qm / mm calculations , 
 we used the nwchem program package .
the 
 qm subsystems were treated in the dft/6 - 31 g * * 
 approximation with the functional bb1k designed for simulations of chemical kinetics .
hydrogen capping 
 atoms were added to the truncated bonds on the border of qm and mm 
 regions .
for the total energy difference ; 1.5  10 a.u . and 1.0  10 a.u . for maximal gradient 
 and gradient root - mean - square ; and 6.0  10 a.u . and 4.0  10 a.u . for maximum and 
 root - mean - square cartesian step , respectively
calculations were performed 
 in the electronic embedding scheme without 
 cutoff for electrostatic and van der waals interactions .
calculations 
 with the car  parrinello molecular dynamics 
 with qm / mm potentials for the mmp  oligopeptide complexes were 
 carried out using the quickstep module of the cp2k program package .
forces and energies in the qm subsystems ( constructed similarly 
 to the corresponding qm / mm simulations ) were computed using the density 
 functional theory with the gaussian and plane wave basis sets ( dft / gpw ) 
 approximation .
the blyp functional with 
 the empirical dispersion corrections and 
 the qzv2p basis set with goedecker  teter 
hutter pseudopotentials were applied . the multigrid approach with the cutoff of the finest grid level of 
 350 ry was utilized .
sets of md simulations were performed in the nvt ensemble 
 at t = 300 k with the periodic boundary conditions 
 using the verlet integrator with a time step of 0.5 fs . to compare 
 predictions of the car  parrinello molecular dynamics 
 with qm / mm potentials and those of the classical md
, we also performed 
 trajectory calculations for the mmp9-regasepin1 complex by using namd with the charmm force field parameters .
a theoretical assignment of the protonation state of polar residues 
 requires calculation of pka values of 
 titrable groups in proteins , which presents a difficult task ( see , 
 e.g. , ref ( 29 ) ) .
attempts 
 to use automated procedures such as propka primarily lead to conclusions consistent with a customary assignment , 
 assuming that usually lys and arg are positively charged and glu and 
 asp are negatively charged inside the protein matrix .
we applied this 
 rule at the stage of constructing molecular models and manually checked 
 the hydrogen bond networks in local environments of polar groups : 
 every electronegative atom should be saturated by hydrogen bonds . 

the initial assignment of protons may be altered in simulations if 
 the corresponding molecular groups are included to quantum subsystems .
the specific atoms assigned to the qm parts are shown in figures 13 in the results section , below .
the initial coordinates 
 of the model systems were taken from the corresponding crystal structures 
 specified below for each particular simulation .
the 
 sulfur atom of the cysteine in the prodomain of mmp ( prommp ) has been 
 shown to inactivate the catalytic zinc - containing domain .
after proteolytic activation , a part of prommp , 
 the oligopeptide , which contains a cysteine , simultaneously dissociates 
 from the active site .
we constructed 
 a complex composed of the mmp-9 catalytic domain and the remaining 
 part of prommp , met - arg - thr - pro - arg - cys - gly - val - pro - asp - leu - gly - arg 
 ( inh ) , on the basis of the corresponding crystal structure 
 of prommp-9 ( pdb i d : 1l6j ) .
the inhibitor 
 regasepin1 , pro - arg - cys - bip - cys - gly - glu ( bip stands for biphenylalanine ) , 
 has a primary structure similar to inh but acts as an 
 inhibitor of mmp-9 .
we constructed the mmp-9regasepin1 complex 
 by modifying the amino acids in the mmp-9inh construct .
equilibrium geometry configurations of both complexes 
 were computed using the qm / mm approach . to justify the use of qm(blyp / gpw - qzv2p)/mm(charmm ) 
 approximation in subsequent molecular dynamics simulations , we compared 
 the structures of the mmp-9regasepin1 complex optimized in 
 two approaches : ( qm(bb1k/6 - 31g**)/mm(amber ) and qm(blyp / gpw - qzv2p)/mm(charmm ) . 

the car  parrinello molecular dynamics simulations were 
 initiated from the qm / mm optimized structures . in figure 1
, we show the equilibrium geometry configurations 
 obtained for the mmp-9inh ( panel a ) and mmp-9regasepin1 
 ( panel b ) complexes . here
, the numbering of the amino acid residues 
 in the ligand refers to the structure pdb i d : 1l6j . according to calculation 
 results , the qm / mm optimized minimum energy structures corresponds 
 to the unprotonated cys99protonated glu2 pair .
an initial 
 manual assignment assumed protonated cys99 in the ligand and unrpotonated 
 glu2 in the protein .
both qm / mm and molecular dynamics simulations 
 resulted in the conclusion that his1 is shifted out of the initial 
 coordination shell of zinc in the mmp-9regasepin1 complex , 
 as shown in figure 1 ( panel c ) and table 1 .
table 1 summarizes 
 the key distances obtained 
 in qm / mm optimization and car  parrinello molecular dynamics 
 simulations of mmp-9inh and mmp-9regasepin1 
 complexes . in the mmp-9regasepin1 complex ,
his1 escapes from 
 the coordination sphere of zn , and this shell becomes 
 more flexible ( figure 1c ) , as indicated by 
 the large corresponding standard deviation ( table 1 ) .
the distance between the nitrogen atom of his1 and the 
 zn is shorter in the qm / mm optimization compared with 
 the mean value obtained in md simulations , but both values definitely 
 show the absence of the coordination bond .
other coordination bond 
 lengths are similar , showing no other disturbances in the mmp-9 active 
 site .
additional stabilization of the bound mmp-9regasepin1 
 structure is due to the stacking interactions between the newly introduced 
 biphenylalanine and tyr179 of the catalytic domain .
equilibrium geometry 
 configurations obtained for ( a ) mmp-9inh and 
 ( b ) mmp-9regasepin1 complexes ; distances are 
 shown in angstroms (  ) . for the mmp-9regasepin1 complex , 
 the distances in parentheses were obtained in qm / mm minimization with 
 the blyp / gpw - qzv2p and charmm approaches .
panel c illustrates variations 
 in the distance between the nitrogen atom of his1 and the zn along the md trajectory in the mmp-9regasepin1 complex . 
 here and in figures 2 and 3 ,
the green , blue , red , white , and light blue colors correspond 
 to carbon , nitrogen , oxygen , hydrogen , and zinc atoms , respectively . for the latter , 
 results of 10 
 and 60
rows with md data show the mean 
 values and standard deviations ( in parentheses ) .
as illustrated in figure 1b , his1 is replaced 
 by cys101 from the ligand in the mmp-9regasepin1 complex . 

the metal ion zn has tetrahedral coordination in both 
 cases : in the mmp-9inh complex , zinc forms coordination 
 bonds with three nitrogen atoms of histidines in mmp-9 and with the 
 sulfur of cys99 in inh . in regasepin1
since zn prefers coordinating with the more polarizable sulfur atom 
 ( rather than with nitrogen ) , cys101 substitutes his1 in the zinc shell . 

the newly formed coordination sphere includes two cysteines from the 
 inhibitor and two histidines from the enzyme , leading to the classical , 
 stable cys2his2 zinc finger motif .
importantly , 
 the mean values and standard deviations from md trajectories 
 corresponding to 10 and 60 ps lengths are practically the same ( last 
 rows of table 1 ) , indicating that the structural 
 motif of the zinc coordination sphere is stable . taking into account 
 that running the car  parrinello molecular dynamics with qm / mm 
 potentials and a fairly large quantum subsystem is very expensive , 
 here we can rely on the data from relatively short trajectories initiated 
 from the qm / mm optimized structures . to understand the importance 
 of quantum mechanics - based calculations
, 
 we also performed classical md simulations for the mmp-9-regasepin1 
 complex by using two different sets of partial charges on atoms for 
 the active site .
one set uses the charmm force field parameters , and 
 another set , the electrostatic potential ( esp ) charges , is obtained 
 from the electron density distribution calculated with the bb1k/6 - 31 g * * 
 approximation .
these computational data ( summarized in the supporting information ) demonstrate that the 
 results of classical md strongly depend on the choice of the partial 
 charges on atoms and predict unreasonable structures of the zinc coordination 
 shell .
we started from the known x - ray structure ( pdb 
 i d : 3ayu(33 ) ) of the inhibitor app - ip complexed with the 
 glu121ala mutant of mmp-2 .
this ordering of amino acid residues corresponds 
 to the x - ray structure pdb i d : 3ayu ; however , in figure 2 and the text , we mark this residue as number 2 . to restore 
 the native sequence in mmp-2
, we introduced glu at position 121 ( or 
 position 2 in figure 2 ) and made point mutations 
 in app - ip , aiming to facilitate formation of the zinc finger motif 
 upon complexation with mmp-2 . in the mmp-2app - ip complex , 

we modified this residue 
 to cysteine ( asp6cys ) because its side chain has a similar volume 
 and can perfectly coordinate zn .
we also mutated tyr3cys 
 to introduce an additional residue capable of entering the coordination 
 sphere of zinc , naming the resulting oligopeptide app - ip .
equilibrium geometry configurations obtained for the ( a ) mmp-2app - ip 
 and ( b ) mmp-2app - ip complexes . graphs showing 
 distances (  ) between atoms along md trajectories : ( c ) corresponds 
 to the distances between the nitrogen of his5 and the zn in the mmp-2app - ip complex ; ( d ) corresponds 
 to the distances between asp6od1 ( blue ) or asp6od2 ( red ) and the zn in the mmp-2app - ip complex .
figure 2 shows equilibrium geometry configurations 
 obtained for the mmp-2app - ip and mmp-2app - ip complexes .
zn is coordinated by the asp6 carboxylic 
 group of app - ip and by three histidines from mmp-2 .
the mmp-2app - ip complex has a tetrahedral zn coordination 
 sphere formed by two cysteines from the proposed oligopeptide and 
 two histidines from the enzyme .
rows with md data 
 show the mean 
 values and standard deviations ( in parentheses ) .
substrate 
 ( es ) complex of the 
 mmp-2 catalytic domain with the model substrate , ace - gln - gly  
 ile - ala - gly - nme , obtained previously , and suggested specific modifications to facilitate formation of 
 a zinc finger motif .
we modified the gly  ile scissile fragment 
 with a d - cys  cys dipeptide and constructed a species 
 we named inh .
we chose the d - isomer because we wanted the side chain of the amino acid replacing 
 gly to face toward the active site of the enzyme , and the side chain 
 of the l - isomer would orient away from the active site .
figure 3 shows 
 equilibrium geometry configurations obtained for the es complex and the complex of the mmp-2 catalytic 
 domain and the suggested inhibitor inh . upon binding ,
inh perturbs the 
 coordination sphere of zn and substitutes one of the 
 coordinating histidines ( his5 ) of the enzyme with a cysteine from 
 the oligopeptide because the zn ion prefers to form bonds 
 with the sulfur atom rather than with nitrogen or oxygen .
moreover , d - cys displaces a catalytic water molecule from the active site , 
 thus eliminating the possibility of an enzyme - catalyzed reaction . 

in addition , these amino acid modifications lead to elongation of 
 the coordination bond between the oxygen atom of the peptide group 
 of the oligopeptide and the znion .
this distance is crucial 
 in the es complex because zn polarizes the carbonyl group 
 of the peptide fragment to facilitate nucleophilic attack and helps 
 to stabilize the tetrahedral intermediate formed in the first stage 
 of the hydrolysis . in the enzyme 
equilibrium geometry configurations for ( a ) the es complex 
 of mmp-2 
 with a natural substrate and ( b ) the complex of mmp-2 with inh .
although 
 exploring properties of the well - known zn(cysxhis4-x ) structural 
 pattern in proteins continues to attract attention ( e.g. , see recent 
 papers ) , to the best of our knowledge , the idea of utilizing 
 formation of the zinc finger motif upon mmp  ligand complexation 
 to assemble oligopeptide - based mmp inhibitors is presented here for 
 the first time .
we use modern molecular modeling tools , including 
 the qm / mm method , to support such a hypothesis .
other qm / mm - based 
 theoretical approaches to model ligand binding sites of mmp-9 beyond 
 the zinc finger construct are described in the literature .
the mmp catalytic motif is composed of three permanent zn ligands ( his ) and a catalytic water molecule , which is very 
 labile . 

usually , inhibitors based on small - molecule organic compounds provide only one additional ( albeit more stable ) coordination bond 
 over that from the reactive water molecule . in the strategy described 
 in this work , two additional coordination bonds , arising from the 
 ligand , can be introduced to the catalytic zinc . the zn(cys2his2 ) pattern formed in the enzyme 
ligand 
 complex in place of an initial zn(his3 ) construct 
 in the enzyme active site should provide additional stabilization 
 to the complex and therefore enhance the 
 inhibition power of a peptidomimetic .
correspondingly , introducing 
 cysteine residues into the substrate fragment responsible for binding 
 with the zinc - containing pocket favors production of mmp inhibitors . 

keeping the other residues found in natural substrates that are responsible 
 for recognizing binding sites in the enzyme should enhance the specificity 
 of the ligand . in this work
, we examined this strategy using 
 three different oligopeptide 
 systems : ( i ) the mmp-9 prodomain and regasepin1 , ( ii ) app - ip and its 
 derivative , and ( iii ) the natural substrate and its derivative .
the oligopeptide inhibitor regasepin1 is specific to mmp-9 ( ic50 = 1 m ) ; however , its interaction mechanism has not 
 been characterized .
following up on the original work presented by 
 hu et al . , we found that its sequence 
 is similar to that of the prodomain of mmp-9 . by using the x - ray structure 
 of full - length mmp-9
, we constructed a complex made up of the mmp-9 
 catalytic domain and the part of the prodomain formed after the proteolytic 
 activation reactions .
we found that the mmp-9regasepin1 complex 
 is characterized by a stable zinc finger motif composed of two cysteines 
 from the inhibitor and two histidines from the enzyme .
this explains 
 the dramatic differences in the binding 
 properties of inh , which spontaneously dissociates from 
 mmp-9 and regasepin1 .
the main structural drawback of regasepin1 is 
 that it has a twisted loop structure , and therefore , it interacts 
 modestly with the binding sites of the catalytic domain . according 
 to the x - ray data
, the latter problem can be resolved 
 for another oligopeptide inhibitor , app - ip .
it binds tightly to the 
 mmp-2 catalytic domain and is located in the channel formed by the 
 amino acids of the binding sites , which explains its low ic50 value of 0.03 m .
armed with the knowledge of the regasepin1 
 binding mechanism , we proposed a mutated variant of app - ip capable 
 of forming a cys2his2 zinc finger motif upon 
 complexation with mmp-2 .
the suggested point mutations do not affect 
 binding properties of the peptidomimetic , but provide additional attractive 
 interactions with zn : app - ip coordinates the znion via the carboxylate group , whereas app - ip does 
 this via two thiolate groups .
it should be noted that both app - ip 
 and regasepin1 are oriented 
 in the opposite n - to - c direction compared with the natural substrates . 

such an arrangement disturbs the reactive configuration of the active 
 site , displacing a catalytic water molecule in particular . to the 
 best of our knowledge ,
neither structural data nor other evidence 
 of oligopeptide - based inhibitors that bind to mmp in a direction similar 
 to the native substrate has been reported .
we suppose that the main 
 reason for this is that mmp binding sites are constructed in such 
 a manner that a substrate binds in a specific way .
namely , the side 
 chain of the amino acid immediately preceding the scissile bond is 
 oriented to the solution , whereas the next one is oriented toward 
 the enzyme .
this provides the free volume in the active site that 
 is occupied by the catalytic water molecule .
correspondingly , we suggested 
 introducing d - cys before the scissile fragment in the substrate . 

its side chain orients toward the active site of mmp , opposite to 
 the l - form s orientation , pushing out a catalytic 
 water molecule and coordinating the zn instead .
another 
 cys was introduced to the sequence of the natural substrate to help 
 create the zinc finger motif .
the presence of a d - amino acid 
 in the oligopeptide has an additional advantage in that this inhibitor 
 should be more resistant to other proteases in in vivo applications .
we observed similar behavior by the side chains of key amino acids 
 in all structures of mmp with inhibitors capable of forming the zinc 
 finger motif . upon binding , one of the introduced cysteines substitutes 
 the histidine from the coordination sphere .
figures 13 show that it can be either 
 his1 or his5 , depending on the direction of the cysteine attack .
regasepin1 complex , cys101 substitutes his1 from 
 the coordination sphere to form a new zn tetrahedral 
 coordination . for app - ip and inh , cys3 and cys ,
encouraged 
 by research showing that complexation of mmp-9 with 
 its inhibitor regasepin1 is accompanied by formation of a structural 
 pattern known as the zinc finger motif , zn(cys2his2 ) , in place of the initial zn(his3 ) construct in the enzyme active site , we suggested a novel 
 strategy to assemble efficient mmp inhibitors on the basis of oligopeptide 
 ligands .
the approach was to introduce cysteine residues into appropriate 
 places in natural substrates while keeping their native outermost 
 amino acid residues to guarantee specific binding to the enzyme .
exploring 
 this hypothesis , we constructed , in silico , a more promising variant 
 of the mmp-2 inhibitor app - ip by considering two replacements in the 
 substrate : tyr3cys and asp6cys .
molecular modeling shows that this 
 peptide variant blocks an enzyme active site via the zn(cys2his2 ) zinc finger construct .
a natural 
 substrate of mmp-2 , ace - gln - gly  ile - ala - gly - nme , can also 
 be converted to an inhibiting compound by two replacements , ile by 
 cys and gly , by the d - isomer of cys , thus favoring formation 
 of the zinc finger motif .
 discovering ways to control the activity 
 of matrix metalloproteinases 
 ( mmps ) , zinc - dependent enzymes capable of degrading extracellular 
 matrix proteins , is an important field of cancer research . 
 we report 
 here a novel strategy for assembling mmp inhibitors on the basis of 
 oligopeptide ligands by exploring the pattern known as the zinc finger 
 motif . 
 advanced molecular modeling tools were used to characterize 
 the structural binding motifs of experimentally tested mmp inhibitors , 
 as well as those of newly proposed peptidomimetics , in their zinc - containing 
 active sites . 
 the results of simulations based on the quantum mechanics / molecular 
 mechanics ( qm / mm ) approach and car  parrinello molecular dynamics 
 with qm / mm potentials demonstrate that , upon binding of regasepin1 , 
 a known mmp-9 inhibitor , the zn2+(his3 ) structural 
 element is rearranged to the zn2+(cys2his2 ) zinc finger motif , in which two cys residues are borrowed 
 from the ligand . following consideration of the crystal structure 
 of mmp-2 with its inhibitor , the oligopeptide app - ip , we proposed 
 a new peptidomimetic with two replacements in the substrate , tyr3cys 
 and asp6cys . 
 simulations show that this peptide variant blocks an 
 enzyme active site by the zn2+(cys2his2 ) zinc finger construct . 
 similarly , a natural substrate of mmp-2 , 
 ace - gln - gly  ile - ala - gly - nme , can be converted to an inhibiting 
 compound by two replacements , ile by cys and gly by the d isomer of cys , favoring formation of the zinc finger motif . 
Introduction
Materials and Methods
Results
Discussion
Conclusion