BACKGROUND
serine proteases play an important role in processes such as blood clotting, digestion and in some pathways of cell development  <cit> .
serine proteases can hydrolyze either peptide bonds or esters.
proteases digest proteins by hydrolyzing the peptide bonds which are responsible for keeping amino acids together  <cit> .
the cleavage specificity of elastase, trypsin, chymotrypsin and other serine proteases depends on the volume/size, form/shape, and polarity/charge/hydrophobicity of the specific part of a protein surface where a substrate will be docking - the specificity pocket  <cit> .
there are three amino acid residues responsible for the enzymatic activity that are present in all serine proteases, which are denominated as the catalytic triad: his  <dig>  asp  <dig> and ser  <dig> .
interestingly, out of those three amino acids, only asp  <dig> does not make part of the interface .
this is due to the fact that asp  <dig> is already not accessible to solvent in isolated enzyme, because this amino acid is located at the very bottom of the active site cleft where solvent molecules  can not access it.
the role of the first two amino acids  during trypsin catalysis, for example, is to function as a proton shuttle.
trypsin cleaves peptides after lys and arg residues with the co-participation of asp  <dig>  which interacts with the positive charge on peptide  <cit> .
chymotrypsin, on the other hand, cleaves proteins after aromatic  residues  <cit> .
to achieve such specificity, in chymotrypsin, one can easily identify the existence of the hydrophobic pocket, normally shielded by met  <dig>  thrombin is a protease which cleaves peptides with more specificity than trypsin: it requires arg on "p1" position  <cit> .
our main objective in this work is to expand the scope of studies that analyze enzyme specificity by including into observations not only the catalytic triad and binding pocket but also a wider category of amino acids which we have named the interface forming residues   <cit> .
namely, only a part of the molecular surface is shielded from solvent upon formation of a bi-molecular complex.
the residues having a lower accessibility to solvent upon complex formation have an important role in the process of docking and also in defining specificity  <cit> .
therefore, we were motivated to calculate which amino acids are shielded from solvent in the bi-molecular complexes involving serine proteases and different types of inhibitors and then create a table  of all amino acid positions at the interface and their respective occupancies.
by mapping those amino acids as a specific ifr, we are now able to analyze characteristics of each position; and by doing so, we also can make position-specific alignment among different subfamilies of serine proteases.
the key step we needed to solve during our procedural approach was to find a sufficient number of pdb  <cit>  structures containing complexes of serine proteases with respective inhibitors.
it became clear that we would need to either produce those by some novel method or abandon our work because there were not enough samples available in the pdb.
the solution to this challenge is presented in details in materials and methods.
RESULTS
interface forming residue  tables
in order to provide a sufficient volume of data for analysis of the interfaces around active sites of serine proteases  we needed to employ the in silico approach for building corresponding complexes.
the key feature of our work is mapping the ifr 3d profile into a 2d matrix--from a known enzyme-inhibitor structure to those with no known structure for such complex.
mapping is done after structurally aligning all serine proteases with non-redundant sequences.
after selection of  <dig> serine protease structural files , named here as a secondary datamart, and three prototype complexes, containing inhibitor/serine protease complexes, we ended up with total of  <dig> structures with non redundant enzyme sequences.
the list of all corresponding pdb ids for serine proteases used in this work is presented in the table  <dig>  all selected  <dig> serine proteases were then structurally aligned.
upon rigid body docking of the three different inhibitors to  <dig> serine proteases from the secondary datamart, we constructed complexes which, counting also the three prototype ones, totalled to three sets of  <dig> two-chain macromolecules.
the interfaces for those three sets were calculated using surfv program  <cit>  and for each in silico generated complex, we mapped all of the sequence positions that belonged to ifr ensemble into the table shown at figure  <dig>
the last three pdb ids  contain a description of the native complexes of: anionic trypsinogen with bpti, collagenase with ecotin and alpha-chymotrypsin with ovomucoid, respectively.
analysis of the tabulated ifr data  clearly indicates subfamily differences with respect to characteristics, position and frequency of occupancy of interface residues.
the data compiled here can be used as an additional tool for characterizing function and specificity.
this is particularly relevant for planning structure/sequence changes intended to alter protein activity, e.g., from trypsin to chymotrypsin type activity.
more generally, one subfamily type of serine protease can be adjusted to behave like another if corresponding changes are made in an initial protease sequence.
attempts have been made to alter protein activity  <cit> , but they have focused on a single or, at most,  <dig> to <dig> residues.
our data, shown on figure  <dig>  indicate that such alterations can be achieved by eliminating key differences in ifr occupancy.
in case of trypsin, chymotrypsin, elastase and thrombin, each bound to bpti, differences are present at  <dig> ifr positions .
in the table shown at figure  <dig>  for instance, sequence position  <dig> is occupied by his  or hydrophobic residues .
position  <dig>  is occupied by mostly polar residues .
positions  <dig>   <dig> and  <dig> are occupied by mostly polar residues in at least  <dig> subfamilies.
in general, positions were designated as "ph" if they were occupied by mostly  polar and occasionally some hydrophobic residues.
similarly, ifr positions occupied by only charged residues were marked as "c", those with only polar residues as "p" and those occupied by only hydrophobic residues as "h".
a combination of letters would mean presence of residues belonging to multiple/distinct amino acid classes.
capital letters are used to designate a predominant residue presence  while small letters designate an occasional presence.
there are less restrictive positions which can be occupied by different classes of amino acids in either one sub family  or across all subfamilies .
a specific analysis of the position  <dig>  shows that it is less restrictive for trypsins  and elastases  than for chymotrypsins  and thrombins .
in order to additionally facilitate analysis of the data at figures  <dig>   <dig> and  <dig>  we have labelled with the gray background color those ifr positions which are populated, within a subfamily, by a single residue type or are absent from the ifr ensemble .
it is important to realize that the presence of one type of amino acid per subfamily is treated here in a same fashion as the absence of that particular position from the ifr ensemble because we understand that the event we call "no-show" for particular position is equally information dense as the information on type of occupancy for particular ifr position.
those ifr positions occupied in each one of four sub families by one predominant type of amino acid, present in at least 50% of serine proteases showing this particular ifr, marked here with the "*", are labelled in light gray.
therefore, the analysis of figures  <dig>   <dig> and  <dig> is aided by four distinct labels: 1) residue "aminochromography", 2) dark gray background, 3) light grey background and 4) "occupancy type".
the fourth label is present in the last column and indicates the two predominant residue classes present in the preceding  <dig> columns .
similar analysis was done for two other inhibitors: bpti with  <dig> identified ifr positions at the enzyme side , and ovomucoid third domain with  <dig> identified ifr positions at the enzyme side .
from the data presented in figures  <dig> and  <dig>  ifr locations  <dig> and  <dig> are shown to contain only polar residues .
by comparing the data from the figures  <dig>   <dig> and  <dig>  the complexes of serine proteases with ecotine inhibitor compile  <dig> restrictive ,  <dig> half-restrictive  and  <dig> non-restrictive ifr positions .
in the case of the bpti and ovomucoid inhibitors, respective data are:  <dig>   <dig> and  <dig> ;  <dig>   <dig> and  <dig> .
those numbers indicate that ecotine binding to serine proteases involves  <dig> % restrictive, 25% less restrictive and  <dig> % non restrictive ifr positions while bpti and ovomucoid show respectively: 39%,  <dig> % and  <dig> %; 32%,  <dig> % and 47%.
consequently, these numbers show that the ovomucoid inhibitor binding to serine proteases is less specific than the binding of ecotine, which in turn is less specific than bpti.
the experimental ki  values reported in the literature for bpti against trypsin is  <dig> × 10- <dig> while for the chymotrypsin it goes down to  <dig> × 10- <dig> and for elastase to  <dig>  × 10- <dig> .
on the other side, turkey ovomucoid third domain was reported to interact with very similar association constant with eight different serine proteinases-bovine chymotrypsins a and b, porcine pancreatic elastase i, proteinase k, streptomyces griseus proteinases a and b, and subtilisin-carlsberg  <cit> .
pál et al.
<cit>  reported the following ki  values for ecotine against trypsin, chymotrypsin and elastase, respectively:  <dig> × 10- <dig>   <dig> × 10- <dig> and  <dig>  × 10- <dig>
amino acid composition at serine protease interfaces
additionally, the difference between the polarity and hydrophobicity of the enzyme surface and interface was investigated for each in silico complex formed.
the buried area and total enzyme free surface area  was compared with respect to the type of residues present.
again, the following residue classes were used: charged, hydrophobic and polar .
the data presented in figure  <dig> indicate that most interfaces, in all serine protease subfamilies bound to either bpti, ecotine or ovomucoid third domain, have a general preference for polar residues .
conversely, most of free enzyme surfaces  show a preference for charged residues  although some  also do show a smaller  preference for hydrophobic residues .
some subfamilies do not follow these patterns.
the interface of the streptogrisin subfamily shows a high presence of glycine residues for complexes with each of the three inhibitors , while the rest of the surface shows preference for polar residues .
elastase shows the lowest differences among interface and surface preferences for  <dig> classes of amino acids defined in this work.
charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily and the three inhibitors.
this prompts speculation about how important this difference in ifr characteristics is for maintaining virulence of those organisms.
the upper part of the figure  <dig> shows a strong presence of hydrophobic residues at the interfaces , while the bottom part of the figure  <dig> shows an absence  of the hydrophobic residues at the interface .
the bottom part of the figure  <dig> shows an increase in the presence of hydrophobic residues at the free enzyme surface area.
the surfaces  have a prevalence of charged residues while the interface contains more polar  residues .
thus, the ifr pocket of serine proteases is not formed by predominantly hydrophobic residues; it is a rather polar environment.
also, the interface contains much fewer  charged residues .
if our data are compared with the ones from janin et.
al.
<cit> , one can observe that the serine proteases are endowed with a specific category of interfaces in terms of the contribution of the  <dig> amino acids types to that area: subfamilies vary in the contribution of hydrophobic residues and are almost constant in having a lower preference for the charged residues, as presented at the table  <dig>  these results point to the fact that although all four serine protease subfamilies do not show a great variability with respect to the percentage of the enzyme total surface area being buried upon in silico complex formation with the bpti, ecotine and ovomucoid third domain , the percentage of the residue classes contributing to the interface varies significantly.
the percentage that charged and polar residues contribute together to the total buried area in trypsin/bpti is 86%, in chymotrypsin/bpti is 65%, in elastase/bpti is 75%, and for thrombin/bpti is 84%.
for all three inhibitors examined, thrombin and trypsin have the highest percentage of charged and polar residues contributing to buried area while elastase and chymotrypsin have the lowest.
the details for the difference in occupancies are presented in figure  <dig> and table  <dig>
the serine proteases are bound to bpti, ecotine and ovomucoid third domain inhibitors.
areas occupied by the three classes of amino acids, with respect to the total buried enzyme area, are expressed as a percentage.
the numbers between the brackets show the number of enzyme - inhibitor complexes included in this analysis for each subfamily.
the column containing polar residue participation also contains  the participation of glycine residues.
those differences may be aligned with the corresponding function that each protease performs.
however, it is important to realize the boundaries we should not cross when applying what we have learned here based on inhibitor-protease binary complexes to substrate-protease complexes.
nevertheless, we assume that inhibitors of serine proteases bind to the active site clefts of their target proteases in a manner that is thought to resemble the binding mode of substrates .
trypsin and thrombin handle the positive charges of a polypeptide by imposing the  charge/polarity of their own ifr surface.
chymotrypsin mitigates the spatial constraints imparted by large hydrophobic  residues present in the substrate/inhibitor by accommodating glycines and hydrophobic residues into its own interface.
elastases, on the other hand, utilize hydrophobic amino acids among their ifrs to complement small, hydrophobic amino acids at the cleavage site of a polypeptide.
the general trends for interfaces described above, when combined with the selectivity of ifr positions discussed earlier in this section, provide a more complete description of the specificity of serine protease subfamilies in both general and detailed way, although, as we expected from the beginning, there is no canonical code that we may convey from this analysis.
benchmarking the accuracy of the "hard docking" procedure for ifr identification
the "confusion matrix" presented at the table  <dig> contains  <dig> categories of evaluators for ifr identification.
the true positives  are ifr residues present in both the native and the rigid body docked complexes.
the true negatives  are solvent exposed residues both in native and in in silico complexes.
the false positives  are residues present in the rigid body docked ifr table but absent in natural complexes and the false negatives  are not buried in native complexes but appear in the ifr of the in silico complexes.
the accuracy was calculated based on this formula: acc = /, where the p =  and n = .
table  <dig> shows these values for 1fy <dig>  and the sixteen complementary complexes.
overall, the accuracy  ranged between  <dig>  and  <dig> .
the true positives  are ifr residues present in both the native and the rigid body docked complexes.
the true negatives  are solvent exposed residues both in native and in in silico complexes.
the false positives  are present in the rigid body docked ifr but absent in natural complexes, and the false negatives  are not buried in native complexes but appear in the ifr in generated in silico complexes.
evaluation of plausibility for in silico generated bi-molecular complexes
the presence of steric clashes involving enzyme and inhibitor residues was analyzed to evaluate our hard docking methodology.
steric clashes could yield unfavourable interactions within complexes that would lead to their decreased stability which might be translated to their faster de complexation in vivo.
we calculated the distances between enzyme and inhibitor residues at the interface for bpti , ecotine  and ovomucoid third domain complexes with serine proteases .
all  <dig> serine proteases are presented using the same numbering  as in table  <dig>  and the same subfamily grouping .
the y-axis shows the distances, measured in Ångstroms, with bars indicating a corresponding standard deviation for each point.
the distances are the average values obtained by calculating the sum of the ca-ca  distances for each amino acid belonging to the ifr ensemble from the enzyme side, to the closest amino acid from the ifr of the inhibitor, and then divided by the number of ifr residues at the enzyme site.
a horizontal line represents the average global mean which was always near  <dig>  Å for all three types of inhibitor complexes.
in a prior paper  <cit> , our research group demonstrated that  <dig>  Å is the cut-off distance that optimizes the isolation, at the residue level, of first order of contacts  in well-packed globular proteins.
the observed  <dig>  Å average global mean distance among cas in enzyme-inhibitor complexes confirms that inhibitors and enzymes form well-packed complexes like those found in the core of globular proteins.
thus, it is plausible to conclude that complexes which present distances less than the average global mean contain spatial clashes.
those complexes can be further submitted to dynamics and minimization in order to rearrange these residues and diminish the steric clashes.
we are set up to try to evaluate how many and which steric clashes would be removed by such procedure, however, this is yet to be done as a next step of our project.
discussion
the data collected, analyzed and presented in this work serves as a tool for understanding binding properties in general and protease specificity in particular, based on interface forming residue  profile alignment.
the procedure we have developed for this work describes a): the ensemble of ifrs for any complex formed between a serine protease and an inhibitor and b): the differences in interface characteristics between various proteases with respect to specificity.
from our data one may not extract a simple, concise and very straightforward rule for separation among proteases in terms of specificity .
nature obviously prepared a mechanism for fine tuning of the activity that yields terms of specificity for each subfamily of serine proteases.
there is obviously some space left for overlaps among ifr characteristics and positions that dictate the specificity for each sub family and consequently, what could have been a simple table with reduced complexity in terms of "colors" and characteristics of amino acids employed at each ifr position, became somewhat blurred.
the data presented here are precise in pointing to such granular/spectral distribution in ifr occupancy that would be directly coupled to the specificity type of studied enzymes.
it is our perception that the beauty of the adaptability of molecular mechanisms, expressed here in form of their specificity, can still be presented in a such simplified way  showing how specificity can be fine-tuned from different points of entry : ifr positions and characteristics of amino acids that occupy those critical positions.
the procedure we described may also open the possibility to yield some details that may account for differences in how strong might be the binding between an enzyme and an inhibitor .
it has been established that increased structural plasticity in the binding pocket increases the variation of substrate size that can fit into the critical space directed toward the catalytic triad which correspondingly decreases the specificity  <cit> .
consequently, it is expected that the higher the stiffness around the binding pocket, the higher the selective pressure will be on a particular substrate.
specificity then can be seen as directly proportional to structural limitations imposed first by the size of the docking space and then by the physical and chemical characteristics of this space.
by focusing our attention on the type of residues occupying the enzyme and inhibitor interfaces, we have set aside the evaluation of how plasticity of binding pocket influences enzyme specificity.
this factor, however, is not taken as a non important one.
in fact, we will be undertaking further examination in order to understand better how plasticity may be accounted for by ifr properties - therefore we will be tackling this task in the future.
as a start, the residue types occupying the interface positions implicitly contain the sort of information which relates directly to plasticity of the site.
CONCLUSIONS
the work presented here offers insight into the structure/function relationship of serine proteases.
the superposition of structurally aligned backbones of serine proteases showed that they use essentially identical scaffolding and achieve a variety of specificities by varying surface residues.
previously, there have been attempts to modify the functionality of serine proteases.
these attempts usually focus on several residues and generally result in the loss of enzyme activity.
our approach explains why and corroborates with the results presented by ma et al.
<cit>  and novozymes biotech, inc.  which patented a technology  by confirming necessity for several ifr amino acids to be either substituted, or deleted  or to be introduced in original protease sequence in order to transform trypsin to chymotrypsin.
based on the results we are reporting here, we are poised to assemble, in the near future, the serine protease superfamily interface data resource as an expanding collection of sequence, structural, and functional information about the serine proteases interface forming residues.
a combination of graphics, images and numerical data will be used to aid in the complete analysis of structure/function relationships that still require our attention before we may fully understand the precise role of each amino acid employed at the particular location by enzymes in order to achieve desired specificity.

 serine proteases play an important role in processes such as blood clotting, digestion and in some pathways of cell development. serine proteases can hydrolyze either peptide bonds or esters. proteases digest proteins by hydrolyzing the peptide bonds which are responsible for keeping amino acids together. the cleavage specificity of elastase, trypsin, chymotrypsin and other serine proteases depends on the volume/size, form/shape, and polarity/charge/hydrophobicity of the specific part of a protein surface where a substrate will be docking - the specificity pocket. there are three amino acid residues responsible for the enzymatic activity that are present in all serine proteases, which are denominated as the catalytic triad: his asp and ser. interestingly, out of those three amino acids, only asp does not make part of the interface. this is due to the fact that asp is already not accessible to solvent in isolated enzyme, because this amino acid is located at the very bottom of the active site cleft where solvent molecules can not access it. the role of the first two amino acids during trypsin catalysis, for example, is to function as a proton shuttle. trypsin cleaves peptides after lys and arg residues with the co-participation of asp which interacts with the positive charge on peptide. chymotrypsin, on the other hand, cleaves proteins after aromatic residues. to achieve such specificity, in chymotrypsin, one can easily identify the existence of the hydrophobic pocket, normally shielded by met thrombin is a protease which cleaves peptides with more specificity than trypsin: it requires arg on "p1" position. our main objective in this work is to expand the scope of studies that analyze enzyme specificity by including into observations not only the catalytic triad and binding pocket but also a wider category of amino acids which we have named the interface forming residues. namely, only a part of the molecular surface is shielded from solvent upon formation of a bi-molecular complex. the residues having a lower accessibility to solvent upon complex formation have an important role in the process of docking and also in defining specificity. therefore, we were motivated to calculate which amino acids are shielded from solvent in the bi-molecular complexes involving serine proteases and different types of inhibitors and then create a table of all amino acid positions at the interface and their respective occupancies. by mapping those amino acids as a specific ifr, we are now able to analyze characteristics of each position; and by doing so, we also can make position-specific alignment among different subfamilies of serine proteases. the key step we needed to solve during our procedural approach was to find a sufficient number of pdb structures containing complexes of serine proteases with respective inhibitors. it became clear that we would need to either produce those by some novel method or abandon our work because there were not enough samples available in the pdb. the solution to this challenge is presented in details in materials and methods.  interface forming residue tables in order to provide a sufficient volume of data for analysis of the interfaces around active sites of serine proteases we needed to employ the in silico approach for building corresponding complexes. the key feature of our work is mapping the ifr 3d profile into a 2d matrix--from a known enzyme-inhibitor structure to those with no known structure for such complex. mapping is done after structurally aligning all serine proteases with non-redundant sequences. after selection of serine protease structural files, named here as a secondary datamart, and three prototype complexes, containing inhibitor/serine protease complexes, we ended up with total of structures with non redundant enzyme sequences. the list of all corresponding pdb ids for serine proteases used in this work is presented in the table all selected serine proteases were then structurally aligned. upon rigid body docking of the three different inhibitors to serine proteases from the secondary datamart, we constructed complexes which, counting also the three prototype ones, totalled to three sets of two-chain macromolecules. the interfaces for those three sets were calculated using surfv program and for each in silico generated complex, we mapped all of the sequence positions that belonged to ifr ensemble into the table shown at figure the last three pdb ids contain a description of the native complexes of: anionic trypsinogen with bpti, collagenase with ecotin and alpha-chymotrypsin with ovomucoid, respectively. analysis of the tabulated ifr data clearly indicates subfamily differences with respect to characteristics, position and frequency of occupancy of interface residues. the data compiled here can be used as an additional tool for characterizing function and specificity. this is particularly relevant for planning structure/sequence changes intended to alter protein activity, e.g., from trypsin to chymotrypsin type activity. more generally, one subfamily type of serine protease can be adjusted to behave like another if corresponding changes are made in an initial protease sequence. attempts have been made to alter protein activity, but they have focused on a single or, at most, to residues. our data, shown on figure indicate that such alterations can be achieved by eliminating key differences in ifr occupancy. in case of trypsin, chymotrypsin, elastase and thrombin, each bound to bpti, differences are present at ifr positions. in the table shown at figure for instance, sequence position is occupied by his or hydrophobic residues. position is occupied by mostly polar residues. positions and are occupied by mostly polar residues in at least subfamilies. in general, positions were designated as "ph" if they were occupied by mostly polar and occasionally some hydrophobic residues. similarly, ifr positions occupied by only charged residues were marked as "c", those with only polar residues as "p" and those occupied by only hydrophobic residues as "h". a combination of letters would mean presence of residues belonging to multiple/distinct amino acid classes. capital letters are used to designate a predominant residue presence while small letters designate an occasional presence. there are less restrictive positions which can be occupied by different classes of amino acids in either one sub family or across all subfamilies. a specific analysis of the position shows that it is less restrictive for trypsins and elastases than for chymotrypsins and thrombins. in order to additionally facilitate analysis of the data at figures and we have labelled with the gray background color those ifr positions which are populated, within a subfamily, by a single residue type or are absent from the ifr ensemble. it is important to realize that the presence of one type of amino acid per subfamily is treated here in a same fashion as the absence of that particular position from the ifr ensemble because we understand that the event we call "no-show" for particular position is equally information dense as the information on type of occupancy for particular ifr position. those ifr positions occupied in each one of four sub families by one predominant type of amino acid, present in at least 50% of serine proteases showing this particular ifr, marked here with the "*", are labelled in light gray. therefore, the analysis of figures and is aided by four distinct labels: 1) residue "aminochromography", 2) dark gray background, 3) light grey background and 4) "occupancy type". the fourth label is present in the last column and indicates the two predominant residue classes present in the preceding columns. similar analysis was done for two other inhibitors: bpti with identified ifr positions at the enzyme side, and ovomucoid third domain with identified ifr positions at the enzyme side. from the data presented in figures and ifr locations and are shown to contain only polar residues. by comparing the data from the figures and the complexes of serine proteases with ecotine inhibitor compile restrictive, half-restrictive and non-restrictive ifr positions. in the case of the bpti and ovomucoid inhibitors, respective data are: and; and. those numbers indicate that ecotine binding to serine proteases involves % restrictive, 25% less restrictive and % non restrictive ifr positions while bpti and ovomucoid show respectively: 39%, % and %; 32%, % and 47%. consequently, these numbers show that the ovomucoid inhibitor binding to serine proteases is less specific than the binding of ecotine, which in turn is less specific than bpti. the experimental ki values reported in the literature for bpti against trypsin is × 10- while for the chymotrypsin it goes down to × 10- and for elastase to × 10-. on the other side, turkey ovomucoid third domain was reported to interact with very similar association constant with eight different serine proteinases-bovine chymotrypsins a and b, porcine pancreatic elastase i, proteinase k, streptomyces griseus proteinases a and b, and subtilisin-carlsberg. pál et al. reported the following ki values for ecotine against trypsin, chymotrypsin and elastase, respectively: × 10- × 10- and × 10- amino acid composition at serine protease interfaces additionally, the difference between the polarity and hydrophobicity of the enzyme surface and interface was investigated for each in silico complex formed. the buried area and total enzyme free surface area was compared with respect to the type of residues present. again, the following residue classes were used: charged, hydrophobic and polar. the data presented in figure indicate that most interfaces, in all serine protease subfamilies bound to either bpti, ecotine or ovomucoid third domain, have a general preference for polar residues. conversely, most of free enzyme surfaces show a preference for charged residues although some also do show a smaller preference for hydrophobic residues. some subfamilies do not follow these patterns. the interface of the streptogrisin subfamily shows a high presence of glycine residues for complexes with each of the three inhibitors, while the rest of the surface shows preference for polar residues. elastase shows the lowest differences among interface and surface preferences for classes of amino acids defined in this work. charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily and the three inhibitors. this prompts speculation about how important this difference in ifr characteristics is for maintaining virulence of those organisms. the upper part of the figure shows a strong presence of hydrophobic residues at the interfaces, while the bottom part of the figure shows an absence of the hydrophobic residues at the interface. the bottom part of the figure shows an increase in the presence of hydrophobic residues at the free enzyme surface area. the surfaces have a prevalence of charged residues while the interface contains more polar residues. thus, the ifr pocket of serine proteases is not formed by predominantly hydrophobic residues; it is a rather polar environment. also, the interface contains much fewer charged residues. if our data are compared with the ones from janin et. al., one can observe that the serine proteases are endowed with a specific category of interfaces in terms of the contribution of the amino acids types to that area: subfamilies vary in the contribution of hydrophobic residues and are almost constant in having a lower preference for the charged residues, as presented at the table these results point to the fact that although all four serine protease subfamilies do not show a great variability with respect to the percentage of the enzyme total surface area being buried upon in silico complex formation with the bpti, ecotine and ovomucoid third domain, the percentage of the residue classes contributing to the interface varies significantly. the percentage that charged and polar residues contribute together to the total buried area in trypsin/bpti is 86%, in chymotrypsin/bpti is 65%, in elastase/bpti is 75%, and for thrombin/bpti is 84%. for all three inhibitors examined, thrombin and trypsin have the highest percentage of charged and polar residues contributing to buried area while elastase and chymotrypsin have the lowest. the details for the difference in occupancies are presented in figure and table the serine proteases are bound to bpti, ecotine and ovomucoid third domain inhibitors. areas occupied by the three classes of amino acids, with respect to the total buried enzyme area, are expressed as a percentage. the numbers between the brackets show the number of enzyme - inhibitor complexes included in this analysis for each subfamily. the column containing polar residue participation also contains the participation of glycine residues. those differences may be aligned with the corresponding function that each protease performs. however, it is important to realize the boundaries we should not cross when applying what we have learned here based on inhibitor-protease binary complexes to substrate-protease complexes. nevertheless, we assume that inhibitors of serine proteases bind to the active site clefts of their target proteases in a manner that is thought to resemble the binding mode of substrates. trypsin and thrombin handle the positive charges of a polypeptide by imposing the charge/polarity of their own ifr surface. chymotrypsin mitigates the spatial constraints imparted by large hydrophobic residues present in the substrate/inhibitor by accommodating glycines and hydrophobic residues into its own interface. elastases, on the other hand, utilize hydrophobic amino acids among their ifrs to complement small, hydrophobic amino acids at the cleavage site of a polypeptide. the general trends for interfaces described above, when combined with the selectivity of ifr positions discussed earlier in this section, provide a more complete description of the specificity of serine protease subfamilies in both general and detailed way, although, as we expected from the beginning, there is no canonical code that we may convey from this analysis. benchmarking the accuracy of the "hard docking" procedure for ifr identification the "confusion matrix" presented at the table contains categories of evaluators for ifr identification. the true positives are ifr residues present in both the native and the rigid body docked complexes. the true negatives are solvent exposed residues both in native and in in silico complexes. the false positives are residues present in the rigid body docked ifr table but absent in natural complexes and the false negatives are not buried in native complexes but appear in the ifr of the in silico complexes. the accuracy was calculated based on this formula: acc = /, where the p = and n =. table shows these values for 1fy and the sixteen complementary complexes. overall, the accuracy ranged between and. the true positives are ifr residues present in both the native and the rigid body docked complexes. the true negatives are solvent exposed residues both in native and in in silico complexes. the false positives are present in the rigid body docked ifr but absent in natural complexes, and the false negatives are not buried in native complexes but appear in the ifr in generated in silico complexes. evaluation of plausibility for in silico generated bi-molecular complexes the presence of steric clashes involving enzyme and inhibitor residues was analyzed to evaluate our hard docking methodology. steric clashes could yield unfavourable interactions within complexes that would lead to their decreased stability which might be translated to their faster de complexation in vivo. we calculated the distances between enzyme and inhibitor residues at the interface for bpti, ecotine and ovomucoid third domain complexes with serine proteases. all serine proteases are presented using the same numbering as in table and the same subfamily grouping. the y-axis shows the distances, measured in Ångstroms, with bars indicating a corresponding standard deviation for each point. the distances are the average values obtained by calculating the sum of the ca-ca distances for each amino acid belonging to the ifr ensemble from the enzyme side, to the closest amino acid from the ifr of the inhibitor, and then divided by the number of ifr residues at the enzyme site. a horizontal line represents the average global mean which was always near Å for all three types of inhibitor complexes. in a prior paper, our research group demonstrated that Å is the cut-off distance that optimizes the isolation, at the residue level, of first order of contacts in well-packed globular proteins. the observed Å average global mean distance among cas in enzyme-inhibitor complexes confirms that inhibitors and enzymes form well-packed complexes like those found in the core of globular proteins. thus, it is plausible to conclude that complexes which present distances less than the average global mean contain spatial clashes. those complexes can be further submitted to dynamics and minimization in order to rearrange these residues and diminish the steric clashes. we are set up to try to evaluate how many and which steric clashes would be removed by such procedure, however, this is yet to be done as a next step of our project. discussion the data collected, analyzed and presented in this work serves as a tool for understanding binding properties in general and protease specificity in particular, based on interface forming residue profile alignment. the procedure we have developed for this work describes a): the ensemble of ifrs for any complex formed between a serine protease and an inhibitor and b): the differences in interface characteristics between various proteases with respect to specificity. from our data one may not extract a simple, concise and very straightforward rule for separation among proteases in terms of specificity. nature obviously prepared a mechanism for fine tuning of the activity that yields terms of specificity for each subfamily of serine proteases. there is obviously some space left for overlaps among ifr characteristics and positions that dictate the specificity for each sub family and consequently, what could have been a simple table with reduced complexity in terms of "colors" and characteristics of amino acids employed at each ifr position, became somewhat blurred. the data presented here are precise in pointing to such granular/spectral distribution in ifr occupancy that would be directly coupled to the specificity type of studied enzymes. it is our perception that the beauty of the adaptability of molecular mechanisms, expressed here in form of their specificity, can still be presented in a such simplified way showing how specificity can be fine-tuned from different points of entry: ifr positions and characteristics of amino acids that occupy those critical positions. the procedure we described may also open the possibility to yield some details that may account for differences in how strong might be the binding between an enzyme and an inhibitor. it has been established that increased structural plasticity in the binding pocket increases the variation of substrate size that can fit into the critical space directed toward the catalytic triad which correspondingly decreases the specificity. consequently, it is expected that the higher the stiffness around the binding pocket, the higher the selective pressure will be on a particular substrate. specificity then can be seen as directly proportional to structural limitations imposed first by the size of the docking space and then by the physical and chemical characteristics of this space. by focusing our attention on the type of residues occupying the enzyme and inhibitor interfaces, we have set aside the evaluation of how plasticity of binding pocket influences enzyme specificity. this factor, however, is not taken as a non important one. in fact, we will be undertaking further examination in order to understand better how plasticity may be accounted for by ifr properties - therefore we will be tackling this task in the future. as a start, the residue types occupying the interface positions implicitly contain the sort of information which relates directly to plasticity of the site.  the work presented here offers insight into the structure/function relationship of serine proteases. the superposition of structurally aligned backbones of serine proteases showed that they use essentially identical scaffolding and achieve a variety of specificities by varying surface residues. previously, there have been attempts to modify the functionality of serine proteases. these attempts usually focus on several residues and generally result in the loss of enzyme activity. our approach explains why and corroborates with the results presented by ma et al. and novozymes biotech, inc. which patented a technology by confirming necessity for several ifr amino acids to be either substituted, or deleted or to be introduced in original protease sequence in order to transform trypsin to chymotrypsin. based on the results we are reporting here, we are poised to assemble, in the near future, the serine protease superfamily interface data resource as an expanding collection of sequence, structural, and functional information about the serine proteases interface forming residues. a combination of graphics, images and numerical data will be used to aid in the complete analysis of structure/function relationships that still require our attention before we may fully understand the precise role of each amino acid employed at the particular location by enzymes in order to achieve desired specificity. 
it also indicates that the interface forming residues which also determine specificity of serine protease subfamily can not be presented in a canonical way but rather as a matrix of alternative populations of amino acids occupying variety of ifr positions. we were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table of all amino acid positions at the interface as well as their respective occupancies. the table of all amino acid positions at the interface and their respective occupancy is created. in this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the interface forming residues. the ifrs are obtained by "rigid body docking" among structurally aligned, sequence wise non-redundant, serine protease structures with inhibitors: bovine pancreatic trypsin inhibitor, ecotine and ovomucoid third domain inhibitor. we propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues for serine proteases and their inhibitors. the serine proteases interfaces prefer polar residues. our work here provides a unique tool for both structure/function relationship analysis as well as a compilation of indicators detailing how the specificity of various serine proteases may have been achieved and/or could be altered. this prompts speculation about how important this difference in ifr characteristics is for maintaining virulence of those organisms. our goal is to establish a platform for analysis of the relationship between ifr characteristics and binding properties/specificity for bi-molecular complexes. therefore, an in silico approach was designed for building corresponding complexes
 enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. in this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the interface forming residues. we were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table of all amino acid positions at the interface as well as their respective occupancies. our goal is to establish a platform for analysis of the relationship between ifr characteristics and binding properties/specificity for bi-molecular complexes.  we propose a novel method for describing binding properties and delineating serine proteases specificity by compiling an exhaustive table of interface forming residues for serine proteases and their inhibitors. currently, the protein data bank does not contain all the data that our analysis would require. therefore, an in silico approach was designed for building corresponding complexes the ifrs are obtained by "rigid body docking" among structurally aligned, sequence wise non-redundant, serine protease structures with inhibitors: bovine pancreatic trypsin inhibitor, ecotine and ovomucoid third domain inhibitor. the table of all amino acid positions at the interface and their respective occupancy is created. we also developed a new computational protocol for predicting ifrs for those complexes which were not deciphered experimentally so far, achieving accuracy of at least.  the serine proteases interfaces prefer polar residues. charged residues were found to be uniquely prevalent at the interfaces between the "miscellaneous-virus" subfamily and the three inhibitors. this prompts speculation about how important this difference in ifr characteristics is for maintaining virulence of those organisms. our work here provides a unique tool for both structure/function relationship analysis as well as a compilation of indicators detailing how the specificity of various serine proteases may have been achieved and/or could be altered. it also indicates that the interface forming residues which also determine specificity of serine protease subfamily can not be presented in a canonical way but rather as a matrix of alternative populations of amino acids occupying variety of ifr positions. 