BACKGROUND
matrix metalloproteinases  are members of the large family of zinc-containing endopeptidases and are biologically attractive drug targets owing to their involvement in tissue remodelling and degradation of extracellular matrix  <cit> .
allegedly, interest in mmps was recently prompted by evidence that a number of synthetic inhibitors used for the treatment of various pathological states, such as inflammation, arthritis, and cancer, triggered unbalanced and, to some extent, unexpected responses of certain mmps; in this respect, mmps have been distinguished as targets, anti-targets and counter-targets  <cit> .
mmp catalytic domains possess high sequence similarity  with a common residue motif, hexghxxgxxh, incorporating  <dig> histidines that coordinate the catalytic zinc ion.
all protein structures exhibit the characteristic fold of zinc-dependent endopeptidases consisting of a five-stranded beta sheet  and three alpha helices.
shaped as a cavity crossing the entire enzyme, the active site is characterized by a number of subsites  <cit>  directly involved in the interaction with physiological substrates and natural or synthetic inhibitors.
the human genome sequence has enabled us to characterize the entire mmp family, a gallery of proteases encoded by  <dig> distinct genes.
this family includes the archetypal mmps, the matrylisin, the gelatinases and the convertase-activable mmps  <cit> .
to date, at least  <dig> human mmps are known and diverse efforts for their classification have been made.
in view of this, the development of new analytical strategies enabling the decoding and proper interpretation of information encrypted in protein structures is indeed an open challenge.
among others, cluster analysis is a valuable approach to this end.
clustering deals with the partitioning of a set of n elements into k groups based on a suitable similarity criterion.
as is well known, clustering is generally performed through parametric and non-parametric methods  <cit> .
the parametric algorithms require prior knowledge of the data structure, enabling the formulation of assumptions, such as establishing the number of clusters to be found.
the clustering problem is, thus, converted into an optimization task, as a cost function is minimized in correspondence to the best partition of the data: typical examples are k-means and deterministic annealing.
non-parametric methods represent the optimal strategy when no prior knowledge of potential clusters is available: these methods make few assumptions about the structure of the data.
examples of non-parametric methods are linkage  algorithms, whose output is a dendrogram displaying the complete hierarchy of clustering solutions on different scales.
a recently proposed non-parametric method is chaotic map clustering   <cit> .
this algorithm was inspired by a study of the statistical properties of chaotic physical systems which are exploited to obtain an optimal partition of data.
the cmc has already been successfully applied to cluster data in different fields, from medicine to engineering and finance; examples are: the detection of buried land mines using dynamic infrared imaging  <cit> ; the study of human evolution by clustering mitochondrial dna sequences  <cit> ; the analysis of electroencephalographic signals to recognize huntington's disease  <cit> ; and the clustering of dow jones stock market companies for portfolio optimization strategies  <cit> .
in the present investigation, cmc was used for the first time to analyse protein structures.
recently analysed through different chemometrical approaches aimed at studying the structural differences  <cit> , the family of mmps was chosen as a case study.
it represented a good benchmark, having a high number of entries in the world wide protein data bank   <cit> .
in this regard, it is worth saying that the cmc algorithm is even more accurate when dealing with large number of data.
mostly based on the electrostatic potential similarity, the present study accounted for a number of mmps higher than previous investigations greatly widening the structural boundaries of the so-called mmp target family landscape  <cit> .
more specifically, previous analyses have been performed on a low number  of mmps to evaluate their selectivity on the basis of grid molecular interaction fields and consensus principal component analyses   <cit> .
other studies, addressing a higher number of mmps , estimated the similarity within the mmp subsites by taking into account ligand interaction energies  <cit> .
based again on grid/cpca, a further analysis has been reported to evaluate mmp selectivity on a larger number of proteins   <cit> .
finally, some of us carried out the screening of all available mmp structures from the pdb and demonstrated that the analysis of the protein sequences enabled us to reproduce the mmp classification based on the structural domain organization  <cit> .
the present analysis of protein electrostatic potential similarities was shown to be effective in obtaining insight into molecular recognition and substrate specificity.
cmc analysis was a successful strategy in landscaping the entire mmp target family as well as in investigating the subsites responsible for molecular selectivity.
despite their diverse fundamentals, the analysis of mmps via cmc provided satisfying results that generally match, or even outperform, those obtainable by applying standard approach such as the unweighted pair group method with arithmetic mean  algorithm.
cmc performances were also challenged to analyse mmp primary structures.
finally, cmc made it possible to properly relate molecular shape similarity of the co-crystallised ligands with void volumes available in the x-ray mmp complexes.
RESULTS
mmp target family landscape
as a first step, electrostatic potential values calculated on the aligned protein structures were analysed using the cmc algorithm to represent the entire mmp family.
in the present analysis, over the course of  <dig> iterations, k was heuristically set at  <dig> while the threshold θ of the mutual information  equal to  <dig>  was chosen since it intercepted the first and flattest plateau of the cluster entropy  yielding the highest member density for the most populated cluster ).
this threshold θ was highlighted through a dashed red line in fig.
<dig> and represents the resolution at which the data are analysed or, in other words, indicates the boundary to consider a given structure as a singleton.
cmc demonstrated high sensitivity in portraying the mmp target family landscape by properly recognising proteins with similar structural motifs among the different mmp subfamily.
when considering domain organization  <cit> , assignment of the mmp structures almost perfectly matched the classification  with the single exception of a gelatinase  that was misplaced in the group of archetypal mmps.
this misallocation was however due to the inclusion of a short peptide, ser187-leu-gly-lys-gly-val <dig>  instead of the three fibronectin modules enabling specific binding with collagen.
this finding emerged also in a previous work concerned with sequence analysis  <cit> .
interestingly, closer analysis revealed that collagenases as well as mt-mmps and stromelysins were properly grouped in full agreement with the classification based on mmp subfamilies.
then, it was observed that mmps of identical class were aggregated into highly homogenous groups, except for two singletons determined by two stromelysins whose x-ray structures  were missing from a stretch of six residues .
the comparison of cmc and upgma  revealed that congruent results were obtained.
however, the dendrogram generated via cmc was indeed more easily interpretable and, to some extent, more informative.
unlike cmc, upgma was in fact unable to generate the classification based on the domain organization  <cit> , which is known as the highest level of mmp classification, but also failed to properly cluster mmp- <dig> and mmp- <dig> .
similarly, the analysis carried out by upgma confirmed that 1qia and 1qic were effectively diverse from the other elements of the same class  and produced a cladogram  with the longest branches for these two proteins.
cmc analyses of mmp binding sites
the second stage of our investigation was focused on mmp active sites and a number of independent cmc analyses were carried out for studying: a) the significant role of the s1' pocket in determining enzyme specificity; b) the residues involved in the s2-s2' stretch embedding the catalytic domain responsible for protein function regulation; c) the region involving s3-s1-s3' subsites constituting a shallow region containing β-strand iv and two slightly variable loops among different mmp isoforms.
a) analysis of the s1' subsite
known as the specificity pocket, the s1' subsite is the most relevant cavity within the mmp active site and is characterised by a loop behind such a pocket  <cit> .
cmc analysis focused on  <dig> residues  whose consensus sequence plyhsftdltrfrlsq, obtained through multiple sequence alignment, disclosed rather lower percentages for most of the amino acids  <cit> .
the study of such a variable residue composition is fundamental for interpreting the structural implications responsible for mmp selectivity.
in this regard, cmc was successful in properly resolving the different mmp classes through variations of the corresponding key-point residues.
as shown in fig.
<dig>  mmps of identical class were grouped into a single subgroup with a high level of similarity and, more importantly, were clearly distinguished from all the others.
interestingly, the assignments obtained through the cmc s1' focused analysis was, again, better interpretable than those attainable via upgma  which failed in grouping the mmp- <dig> and two mmp- <dig> .
however, a closer look at the dendrogram of fig.
<dig> revealed that mt-mmps  formed a single group with mmp- <dig>  such an observation held true also when applying the upgma method.
moreover, it was observed that metallo-elastases were split into two different groups containing  <dig> and  <dig> members, respectively, exhibiting different side-chain conformations of polar residues arg <dig> and lys  <dig> .
similarly, it was observed that the mmp- <dig> cluster required lower similarity values to incorporate the pdb:1mnc crystal protein with a different side-chain conformation of glu <dig> .
in addition, the cmc algorithm designated three structures as singletons.
two of these were stromelysin- <dig> , whereas the third was a collagenase- <dig> .
in particular, the 1c8t structure exhibited a different conformation of the region encompassing leu229-thr-arg-phe-arg <dig> of the loop at the bottom of the s1' pocket despite the consistent overlap of protein backbone atoms.
visual inspection of the pdb:1cqr crystal revealed that it was an apo-form and, unlike other stromelysin- <dig>  presented a more restricted and hampered loop.
interestingly, such an observation was also reported in a recent analysis based on the grid/cpca approach  <cit> .
in addition, the cmc algorithm recorded that residues thr247-gly-lys-ser-his <dig>  were absent in the pdb:1cxv structure that was thus left as a singletons in the dendrogram.
finally, a value of global electrostatic similarity  <cit>  for the s1' subsite was measured applying the following formula:
csim=∑i=1n−1∑j=i+1nsii,jn/ <dig>
where si is the hodgkin index of all the protein pairwise  combinations and n is the total number of protein structures.
being si commutative, csim accounts for n/ <dig> calculations thus avoiding double counts.
the analysis involved the mmp classes with n >  <dig> and resulted in the following decreasing similarity order: mmp- <dig>  > mmp- <dig>  > mmp- <dig>  > mmp- <dig>  > mmp- <dig>  > mmp- <dig>  > mmp- <dig> .
the global electrostatic similarity over all the  <dig> s1' subsites was equal to  <dig> .
these results underlined the relevance of the s1' subsite for molecular selectivity, as higher similarity values were observed within each class  while the similarity among all the classes was significantly lower  .
b) analysis of the s2-s2' subsites
the s2-s2' protein regions represented the catalytic domain and displayed a more pronounced similarity  compared to the s1' subsite.
as expected, this remarkable electrostatic similarity value was directly related to the consistent percentages of residue consensus.
although the detection of electrostatic differences proved even more difficult, cmc was able to perceive the variation of electrostatic potential of diverse polar residues.
encompassing residues from position  <dig> to position  <dig> , the stretch under investigation is the well-known sequence motif, hexghxxgxxh, which is common to all mmps.
as expected, the presence of charged residues among variable residues was immediately detected by the cmc algorithm.
for instance, the presence of negatively charged residues  at position  <dig> implied a consistent variation of the electrostatic potential that was immediately perceived by the cmc algorithm, which resulted in clearly distinct groups including gelatinases, mt-mmps and mmp- <dig>  in addition, the cmc was able to detect the occurrence of different side-chain conformations and even punctual residue mutations.
for instance, gelatinases were split into two groups.
the first collected mmp- <dig> structures incorporating the e402q mutation  while the second group contained the only wild mmp- <dig> structure  and the only x-ray mmp- <dig>  structure.
moreover, mmp- <dig> included a member  of the mmp- <dig> class.
such a crystal structure differed from other mmp- <dig> proteins since it lacked residues  <dig> to  <dig> , whose remaining available space was occupied by the asp <dig> side-chain exhibiting a diverse conformation.
furthermore, the mmp- <dig> group did not include two structures  for the occurrence of the e219a mutation .
interestingly, the upgma method afforded comparable results.
in this regard, the obtained cladogram  associated such elements with clearly distinguishable longer branches emerging from a fairly flat tree-like plot.
c) analysis of the s3-s1-s3' subsites
the residue stretches comprising the s3-s1-s3' subsites disclosed a higher electrostatic similarity  compared to the s1' subsite.
in this regard, the heat map in fig.
<dig>  furnished an immediate idea of the overwhelming dominance of red colours indicating high electrostatic similarity at the s3-s1-s3' subsites.
however, the presence of somewhat limited green-like coloured regions was observed for similarity values lower than  <dig> .
by zeroing values greater than  <dig> , a spy plot  was obtained to conveniently illustrate the high internal similarity of gelatinases as well as their remarkable differences compared with other mmps.
nevertheless, it has to be again remarked that such results are in full agreement with those obtained when applying the upgma method.
in this regard, by inspecting the cladogram obtained via upgma , the interested reader can appreciate that the high inter-class similarity is proved by the occurrence of short links among all mmp structures, with the exception of the gelatinases, as discussed above.
a close assessment of the spy plot disclosed a number of other valuable clues.
for instance, the only x-ray structure of mmp- <dig>  was clearly distinguished from all the others; such a result was in agreement with previous investigation  <cit>  based on rmsd analyses.
similarly, the pdb:1cgl crystal belonging to mmp- <dig> displayed a pronounced dissimilarity compared to the other members of the same class for the likely occurrence of induced fit phenomena.
as shown in fig.
<dig>  the asn <dig> residue was shifted away from its location observed in apo-forms of the same structure  for the likely occurrence of a hydrogen bond interaction with the carbonyl group of co-crystallised peptide inhibitor.
in this regard, it is worth saying that similar considerations can be drawn by inspecting the cladogram generated via upgma .
the cmc and upgma methods differed remarkable in assigning the pdb:1you structure which is completely isolated in the cladogram resulting from upgma and is able to join only the pdb:1hv <dig> through a very long link.
in this regard, the cmc assignment seems to be more interpretable since the pdb:1you, which belongs to mmp- <dig>  is inserted in the cluster of the mmp- <dig> and is directly joined to two of them .
interestingly, the inspection of the pdb:1you structure revealed that its tyr <dig> residue was differently oriented with respect to the other mmp- <dig> and was instead closer to the pdb:2pjt and pdb:1ztq.
cmc analysis of mmp primary structures
cmc was also challenged to evaluate its ability to group proteins on the basis of their sequence similarity.
pairwise sequence distances were represented through a heat map .
as can be seen, high similarity was generally measured in each class.
nevertheless, some sequences  did not meet this trend and thus the cmc algorithm effectively detected them as singletons despite their proximity to other proteins of the same type.
moreover, it was observed that mmp- <dig> and mmp- <dig> were both split into two different subclusters.
this was in part due to the occurrence of punctual mutations  or missing residues so that sequences belonging to two separate clusters were similar at  <dig> %.
ligand analysis via molecular shape similarity
the cmc algorithm was finally used to analyse the molecular shapes of the  <dig> co-crystallised ligand molecules extracted from the pool of the  <dig> examined x-ray mmp structures.
the present analysis was aimed at evaluating binding specificity towards mmps on the basis of the complementarity between void volumes within the mmp binding sites and the molecular shapes of the co-crystallised inhibitors.
specifically, cmc made it possible to relate molecular shape similarity with even subtle diversity of mmp physicochemical environments based on the fundamental assumption that two ligand molecules would have the same shape if their volumes matched exactly.
the cmc algorithm allowed us to obtain a dendrogram encoding various informative levels .
firstly, the co-crystallised inhibitors were assigned according to the their chemical structure, since the observed clusters contain structurally similar inhibitors .
secondly, the cmc was able to discern zinc chelating inhibitors from non-zinc chelating inhibitors regardless of mmp class.
the vast majority of reported mmp inhibitors are zinc chelating compounds, whose x-ray structures reveal that inhibitor binding interactions typically include coordination of the catalytic zinc ion and occupancy of the s1' pocket with a hydrophobic group  <cit> .
however, these are broad spectrum inhibitors lacking selectivity.
in the development of new therapeutic agents targeting mmps, there remains considerable room for new structural classes not coordinating the active site zinc atom  <cit> .
in this regard, exploiting the structural differences between the mmp subtypes, currently an important issue in this area, should lead to more specific inhibitors, intended to avoid the toxic side effects supposedly linked to broad spectrum inhibitors  <cit> .
thirdly, low molecular selectivity was immediately inferred by observing a high concentration of replicates  within the same cluster, although these co-crystallised with different mmps.
finally, the analysis of ligand molecular shapes via cmc algorithm made it possible to gain insight into selectivity beyond the backbone on the basis of the size  of the s1' pocket  <cit> .
inhibitors with bulkier moieties -benzenesulfonylamino and 4'--biphenyl-4-sulfonylamino substituents) were effectively comprised in the same cluster intercepting deep pocket mmp complexes .
CONCLUSIONS
the main objective of the present investigation was to apply the chaotic map algorithm to clustering mmp structures.
based on electrostatic potential values, cmc analyses afforded a comprehensive representation of the intimate relationships existing among mmps, showing that structural differences between proteins reflect genuine variation of their electrostatic potentials.
in particular, cmc analysis of entire mmp structures was successful in accurately reproducing the canonical classification of mmps normally based on domain organization.
such a result was not attained when the analysis was repeated by using the upgma approach.
in addition, cmc demonstrated high sensitivity in discerning even smaller protein stretches, and defining relevant areas in proximity to the binding site.
more importantly, cmc was able to properly detect the variance of electrostatic potential occurring for even punctual mutations of ionisable residues.
furthermore, cmc demonstrated an outstanding aptitude for capturing local distortions of the electrostatic potential probably related to physical incorporation of small ligands inducing smaller structural protein rearrangements.
interestingly, cmc represented a valid strategy even for standard analyses as those involving only mmp sequences.
similarly, cmc was successful in correctly relating the molecular shapes of ligand molecules to the void volumes available within the mmp binding sites.
in this view, cmc could represent a valuable alternative approach or a complement to other clustering methods assessing the structural similarity within protein families.
cmc demonstrated performances comparable to those of the upgma, with the former leading however to more easily interpretable results.
incidentally, it should be said that cmc is tailored to deal with large amounts, and could also have potential in the database mining.

 matrix metalloproteinases are members of the large family of zinc-containing endopeptidases and are biologically attractive drug targets owing to their involvement in tissue remodelling and degradation of extracellular matrix. allegedly, interest in mmps was recently prompted by evidence that a number of synthetic inhibitors used for the treatment of various pathological states, such as inflammation, arthritis, and cancer, triggered unbalanced and, to some extent, unexpected responses of certain mmps; in this respect, mmps have been distinguished as targets, anti-targets and counter-targets. mmp catalytic domains possess high sequence similarity with a common residue motif, hexghxxgxxh, incorporating histidines that coordinate the catalytic zinc ion. all protein structures exhibit the characteristic fold of zinc-dependent endopeptidases consisting of a five-stranded beta sheet and three alpha helices. shaped as a cavity crossing the entire enzyme, the active site is characterized by a number of subsites directly involved in the interaction with physiological substrates and natural or synthetic inhibitors. the human genome sequence has enabled us to characterize the entire mmp family, a gallery of proteases encoded by distinct genes. this family includes the archetypal mmps, the matrylisin, the gelatinases and the convertase-activable mmps. to date, at least human mmps are known and diverse efforts for their classification have been made. in view of this, the development of new analytical strategies enabling the decoding and proper interpretation of information encrypted in protein structures is indeed an open challenge. among others, cluster analysis is a valuable approach to this end. clustering deals with the partitioning of a set of n elements into k groups based on a suitable similarity criterion. as is well known, clustering is generally performed through parametric and non-parametric methods. the parametric algorithms require prior knowledge of the data structure, enabling the formulation of assumptions, such as establishing the number of clusters to be found. the clustering problem is, thus, converted into an optimization task, as a cost function is minimized in correspondence to the best partition of the data: typical examples are k-means and deterministic annealing. non-parametric methods represent the optimal strategy when no prior knowledge of potential clusters is available: these methods make few assumptions about the structure of the data. examples of non-parametric methods are linkage algorithms, whose output is a dendrogram displaying the complete hierarchy of clustering solutions on different scales. a recently proposed non-parametric method is chaotic map clustering. this algorithm was inspired by a study of the statistical properties of chaotic physical systems which are exploited to obtain an optimal partition of data. the cmc has already been successfully applied to cluster data in different fields, from medicine to engineering and finance; examples are: the detection of buried land mines using dynamic infrared imaging; the study of human evolution by clustering mitochondrial dna sequences; the analysis of electroencephalographic signals to recognize huntington's disease; and the clustering of dow jones stock market companies for portfolio optimization strategies. in the present investigation, cmc was used for the first time to analyse protein structures. recently analysed through different chemometrical approaches aimed at studying the structural differences, the family of mmps was chosen as a case study. it represented a good benchmark, having a high number of entries in the world wide protein data bank. in this regard, it is worth saying that the cmc algorithm is even more accurate when dealing with large number of data. mostly based on the electrostatic potential similarity, the present study accounted for a number of mmps higher than previous investigations greatly widening the structural boundaries of the so-called mmp target family landscape. more specifically, previous analyses have been performed on a low number of mmps to evaluate their selectivity on the basis of grid molecular interaction fields and consensus principal component analyses. other studies, addressing a higher number of mmps, estimated the similarity within the mmp subsites by taking into account ligand interaction energies. based again on grid/cpca, a further analysis has been reported to evaluate mmp selectivity on a larger number of proteins. finally, some of us carried out the screening of all available mmp structures from the pdb and demonstrated that the analysis of the protein sequences enabled us to reproduce the mmp classification based on the structural domain organization. the present analysis of protein electrostatic potential similarities was shown to be effective in obtaining insight into molecular recognition and substrate specificity. cmc analysis was a successful strategy in landscaping the entire mmp target family as well as in investigating the subsites responsible for molecular selectivity. despite their diverse fundamentals, the analysis of mmps via cmc provided satisfying results that generally match, or even outperform, those obtainable by applying standard approach such as the unweighted pair group method with arithmetic mean algorithm. cmc performances were also challenged to analyse mmp primary structures. finally, cmc made it possible to properly relate molecular shape similarity of the co-crystallised ligands with void volumes available in the x-ray mmp complexes.  mmp target family landscape as a first step, electrostatic potential values calculated on the aligned protein structures were analysed using the cmc algorithm to represent the entire mmp family. in the present analysis, over the course of iterations, k was heuristically set at while the threshold θ of the mutual information equal to was chosen since it intercepted the first and flattest plateau of the cluster entropy yielding the highest member density for the most populated cluster ). this threshold θ was highlighted through a dashed red line in fig. and represents the resolution at which the data are analysed or, in other words, indicates the boundary to consider a given structure as a singleton. cmc demonstrated high sensitivity in portraying the mmp target family landscape by properly recognising proteins with similar structural motifs among the different mmp subfamily. when considering domain organization, assignment of the mmp structures almost perfectly matched the classification with the single exception of a gelatinase that was misplaced in the group of archetypal mmps. this misallocation was however due to the inclusion of a short peptide, ser187-leu-gly-lys-gly-val instead of the three fibronectin modules enabling specific binding with collagen. this finding emerged also in a previous work concerned with sequence analysis. interestingly, closer analysis revealed that collagenases as well as mt-mmps and stromelysins were properly grouped in full agreement with the classification based on mmp subfamilies. then, it was observed that mmps of identical class were aggregated into highly homogenous groups, except for two singletons determined by two stromelysins whose x-ray structures were missing from a stretch of six residues. the comparison of cmc and upgma revealed that congruent results were obtained. however, the dendrogram generated via cmc was indeed more easily interpretable and, to some extent, more informative. unlike cmc, upgma was in fact unable to generate the classification based on the domain organization, which is known as the highest level of mmp classification, but also failed to properly cluster mmp- and mmp-. similarly, the analysis carried out by upgma confirmed that 1qia and 1qic were effectively diverse from the other elements of the same class and produced a cladogram with the longest branches for these two proteins. cmc analyses of mmp binding sites the second stage of our investigation was focused on mmp active sites and a number of independent cmc analyses were carried out for studying: a) the significant role of the s1' pocket in determining enzyme specificity; b) the residues involved in the s2-s2' stretch embedding the catalytic domain responsible for protein function regulation; c) the region involving s3-s1-s3' subsites constituting a shallow region containing β-strand iv and two slightly variable loops among different mmp isoforms. a) analysis of the s1' subsite known as the specificity pocket, the s1' subsite is the most relevant cavity within the mmp active site and is characterised by a loop behind such a pocket. cmc analysis focused on residues whose consensus sequence plyhsftdltrfrlsq, obtained through multiple sequence alignment, disclosed rather lower percentages for most of the amino acids. the study of such a variable residue composition is fundamental for interpreting the structural implications responsible for mmp selectivity. in this regard, cmc was successful in properly resolving the different mmp classes through variations of the corresponding key-point residues. as shown in fig. mmps of identical class were grouped into a single subgroup with a high level of similarity and, more importantly, were clearly distinguished from all the others. interestingly, the assignments obtained through the cmc s1' focused analysis was, again, better interpretable than those attainable via upgma which failed in grouping the mmp- and two mmp-. however, a closer look at the dendrogram of fig. revealed that mt-mmps formed a single group with mmp- such an observation held true also when applying the upgma method. moreover, it was observed that metallo-elastases were split into two different groups containing and members, respectively, exhibiting different side-chain conformations of polar residues arg and lys. similarly, it was observed that the mmp- cluster required lower similarity values to incorporate the pdb:1mnc crystal protein with a different side-chain conformation of glu. in addition, the cmc algorithm designated three structures as singletons. two of these were stromelysin-, whereas the third was a collagenase-. in particular, the 1c8t structure exhibited a different conformation of the region encompassing leu229-thr-arg-phe-arg of the loop at the bottom of the s1' pocket despite the consistent overlap of protein backbone atoms. visual inspection of the pdb:1cqr crystal revealed that it was an apo-form and, unlike other stromelysin- presented a more restricted and hampered loop. interestingly, such an observation was also reported in a recent analysis based on the grid/cpca approach. in addition, the cmc algorithm recorded that residues thr247-gly-lys-ser-his were absent in the pdb:1cxv structure that was thus left as a singletons in the dendrogram. finally, a value of global electrostatic similarity for the s1' subsite was measured applying the following formula: csim=∑i=1n−1∑j=i+1nsii,jn/ where si is the hodgkin index of all the protein pairwise combinations and n is the total number of protein structures. being si commutative, csim accounts for n/ calculations thus avoiding double counts. the analysis involved the mmp classes with n > and resulted in the following decreasing similarity order: mmp- > mmp- > mmp- > mmp- > mmp- > mmp- > mmp-. the global electrostatic similarity over all the s1' subsites was equal to. these results underlined the relevance of the s1' subsite for molecular selectivity, as higher similarity values were observed within each class while the similarity among all the classes was significantly lower. b) analysis of the s2-s2' subsites the s2-s2' protein regions represented the catalytic domain and displayed a more pronounced similarity compared to the s1' subsite. as expected, this remarkable electrostatic similarity value was directly related to the consistent percentages of residue consensus. although the detection of electrostatic differences proved even more difficult, cmc was able to perceive the variation of electrostatic potential of diverse polar residues. encompassing residues from position to position, the stretch under investigation is the well-known sequence motif, hexghxxgxxh, which is common to all mmps. as expected, the presence of charged residues among variable residues was immediately detected by the cmc algorithm. for instance, the presence of negatively charged residues at position implied a consistent variation of the electrostatic potential that was immediately perceived by the cmc algorithm, which resulted in clearly distinct groups including gelatinases, mt-mmps and mmp- in addition, the cmc was able to detect the occurrence of different side-chain conformations and even punctual residue mutations. for instance, gelatinases were split into two groups. the first collected mmp- structures incorporating the e402q mutation while the second group contained the only wild mmp- structure and the only x-ray mmp- structure. moreover, mmp- included a member of the mmp- class. such a crystal structure differed from other mmp- proteins since it lacked residues to, whose remaining available space was occupied by the asp side-chain exhibiting a diverse conformation. furthermore, the mmp- group did not include two structures for the occurrence of the e219a mutation. interestingly, the upgma method afforded comparable results. in this regard, the obtained cladogram associated such elements with clearly distinguishable longer branches emerging from a fairly flat tree-like plot. c) analysis of the s3-s1-s3' subsites the residue stretches comprising the s3-s1-s3' subsites disclosed a higher electrostatic similarity compared to the s1' subsite. in this regard, the heat map in fig. furnished an immediate idea of the overwhelming dominance of red colours indicating high electrostatic similarity at the s3-s1-s3' subsites. however, the presence of somewhat limited green-like coloured regions was observed for similarity values lower than. by zeroing values greater than, a spy plot was obtained to conveniently illustrate the high internal similarity of gelatinases as well as their remarkable differences compared with other mmps. nevertheless, it has to be again remarked that such results are in full agreement with those obtained when applying the upgma method. in this regard, by inspecting the cladogram obtained via upgma, the interested reader can appreciate that the high inter-class similarity is proved by the occurrence of short links among all mmp structures, with the exception of the gelatinases, as discussed above. a close assessment of the spy plot disclosed a number of other valuable clues. for instance, the only x-ray structure of mmp- was clearly distinguished from all the others; such a result was in agreement with previous investigation based on rmsd analyses. similarly, the pdb:1cgl crystal belonging to mmp- displayed a pronounced dissimilarity compared to the other members of the same class for the likely occurrence of induced fit phenomena. as shown in fig. the asn residue was shifted away from its location observed in apo-forms of the same structure for the likely occurrence of a hydrogen bond interaction with the carbonyl group of co-crystallised peptide inhibitor. in this regard, it is worth saying that similar considerations can be drawn by inspecting the cladogram generated via upgma. the cmc and upgma methods differed remarkable in assigning the pdb:1you structure which is completely isolated in the cladogram resulting from upgma and is able to join only the pdb:1hv through a very long link. in this regard, the cmc assignment seems to be more interpretable since the pdb:1you, which belongs to mmp- is inserted in the cluster of the mmp- and is directly joined to two of them. interestingly, the inspection of the pdb:1you structure revealed that its tyr residue was differently oriented with respect to the other mmp- and was instead closer to the pdb:2pjt and pdb:1ztq. cmc analysis of mmp primary structures cmc was also challenged to evaluate its ability to group proteins on the basis of their sequence similarity. pairwise sequence distances were represented through a heat map. as can be seen, high similarity was generally measured in each class. nevertheless, some sequences did not meet this trend and thus the cmc algorithm effectively detected them as singletons despite their proximity to other proteins of the same type. moreover, it was observed that mmp- and mmp- were both split into two different subclusters. this was in part due to the occurrence of punctual mutations or missing residues so that sequences belonging to two separate clusters were similar at %. ligand analysis via molecular shape similarity the cmc algorithm was finally used to analyse the molecular shapes of the co-crystallised ligand molecules extracted from the pool of the examined x-ray mmp structures. the present analysis was aimed at evaluating binding specificity towards mmps on the basis of the complementarity between void volumes within the mmp binding sites and the molecular shapes of the co-crystallised inhibitors. specifically, cmc made it possible to relate molecular shape similarity with even subtle diversity of mmp physicochemical environments based on the fundamental assumption that two ligand molecules would have the same shape if their volumes matched exactly. the cmc algorithm allowed us to obtain a dendrogram encoding various informative levels. firstly, the co-crystallised inhibitors were assigned according to the their chemical structure, since the observed clusters contain structurally similar inhibitors. secondly, the cmc was able to discern zinc chelating inhibitors from non-zinc chelating inhibitors regardless of mmp class. the vast majority of reported mmp inhibitors are zinc chelating compounds, whose x-ray structures reveal that inhibitor binding interactions typically include coordination of the catalytic zinc ion and occupancy of the s1' pocket with a hydrophobic group. however, these are broad spectrum inhibitors lacking selectivity. in the development of new therapeutic agents targeting mmps, there remains considerable room for new structural classes not coordinating the active site zinc atom. in this regard, exploiting the structural differences between the mmp subtypes, currently an important issue in this area, should lead to more specific inhibitors, intended to avoid the toxic side effects supposedly linked to broad spectrum inhibitors. thirdly, low molecular selectivity was immediately inferred by observing a high concentration of replicates within the same cluster, although these co-crystallised with different mmps. finally, the analysis of ligand molecular shapes via cmc algorithm made it possible to gain insight into selectivity beyond the backbone on the basis of the size of the s1' pocket. inhibitors with bulkier moieties -benzenesulfonylamino and 4'--biphenyl-4-sulfonylamino substituents) were effectively comprised in the same cluster intercepting deep pocket mmp complexes.  the main objective of the present investigation was to apply the chaotic map algorithm to clustering mmp structures. based on electrostatic potential values, cmc analyses afforded a comprehensive representation of the intimate relationships existing among mmps, showing that structural differences between proteins reflect genuine variation of their electrostatic potentials. in particular, cmc analysis of entire mmp structures was successful in accurately reproducing the canonical classification of mmps normally based on domain organization. such a result was not attained when the analysis was repeated by using the upgma approach. in addition, cmc demonstrated high sensitivity in discerning even smaller protein stretches, and defining relevant areas in proximity to the binding site. more importantly, cmc was able to properly detect the variance of electrostatic potential occurring for even punctual mutations of ionisable residues. furthermore, cmc demonstrated an outstanding aptitude for capturing local distortions of the electrostatic potential probably related to physical incorporation of small ligands inducing smaller structural protein rearrangements. interestingly, cmc represented a valid strategy even for standard analyses as those involving only mmp sequences. similarly, cmc was successful in correctly relating the molecular shapes of ligand molecules to the void volumes available within the mmp binding sites. in this view, cmc could represent a valuable alternative approach or a complement to other clustering methods assessing the structural similarity within protein families. cmc demonstrated performances comparable to those of the upgma, with the former leading however to more easily interpretable results. incidentally, it should be said that cmc is tailored to deal with large amounts, and could also have potential in the database mining. 
in this analysis electrostatic potentials are computed and used as input for the cmc algorithm. the cmc algorithm was shown to be a valuable tool in knowledge acquisition and transfer from mmp structures. it was shown that differences between proteins reflect genuine variation of their electrostatic potentials. we describe a pairwise variant of the non-parametric chaotic map clustering algorithm and its application to x-ray mmp structures. the second made it possible to distinguish the mmp classes, demonstrating the high specificity of the s1' pocket, to detect both the occurrence of punctual mutations of ionisable residues and different side-chain conformations that likely account for induced-fit phenomena. matrix metalloproteinases are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. in addition, the analysis has been also extended to analyze the protein primary structures and the molecular shapes of the mmp co-crystallised ligands. finally, cmc was successful for standard pairwise analysis of protein sequences and was used to convincingly explain the complementarity existing between the molecular shapes of the co-crystallised ligand molecules and the accessible mmp void volumes. with this in mind, the perception of the intimate relationships among diverse mmps could be a solid basis for accelerated learning in designing new selective mmp inhibitors. the first investigation resulted in rational figure interpretation of both domain organization as well as of substrate specificity classifications. based on the variation of electrostatic potentials, cmc was successful in analysing the mmp target family landscape and different subsites.
 matrix metalloproteinases are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. with this in mind, the perception of the intimate relationships among diverse mmps could be a solid basis for accelerated learning in designing new selective mmp inhibitors. in this regard, decrypting the latent molecular reasons in order to elucidate similarity among mmps is a key challenge.  we describe a pairwise variant of the non-parametric chaotic map clustering algorithm and its application to x-ray mmp structures. in this analysis electrostatic potentials are computed and used as input for the cmc algorithm. it was shown that differences between proteins reflect genuine variation of their electrostatic potentials. in addition, the analysis has been also extended to analyze the protein primary structures and the molecular shapes of the mmp co-crystallised ligands.  the cmc algorithm was shown to be a valuable tool in knowledge acquisition and transfer from mmp structures. based on the variation of electrostatic potentials, cmc was successful in analysing the mmp target family landscape and different subsites. the first investigation resulted in rational figure interpretation of both domain organization as well as of substrate specificity classifications. the second made it possible to distinguish the mmp classes, demonstrating the high specificity of the s1' pocket, to detect both the occurrence of punctual mutations of ionisable residues and different side-chain conformations that likely account for induced-fit phenomena. in addition, cmc demonstrated a potential comparable to the most popular upgma method that, at present, represents a standard clustering bioinformatics approach. interestingly, cmc and upgma resulted in closely comparable outcomes, but often cmc produced more informative and more easy interpretable dendrograms. finally, cmc was successful for standard pairwise analysis of protein sequences and was used to convincingly explain the complementarity existing between the molecular shapes of the co-crystallised ligand molecules and the accessible mmp void volumes. 