BACKGROUND
matriptase is a type ii transmembrane serine protease of the s <dig> trypsin-like family.
matriptase activity is down-regulated by its physiological inhibitor, hepatocyte growth factor activator inhibitor- <dig>   <cit> .
matriptase is expressed in most epithelial cells and plays essential roles in the establishment and maintenance of epithelial integrity.
new evidence suggests that matriptase is also expressed on mast cells, peripheral blood monocytes and b cells, implicating matriptase in the physiological and pathologic functions of these cells  <cit> .
knock down studies in mice have shown that the protease is important in postnatal survival, epidermal barrier formation, hair follicle growth and thymichomeostasis  <cit> .
at the same time, genetic studies using zebra fish and mice have indicated that the activity of matriptase is critical for tissue-integrity and function, and must be strictly controlled by hai- <dig>  <cit> .
the catalytic domain of matriptase is tethered to the cell surface via its n-terminal signal anchor, linked by a sea urchin sperm protein/enterokinase/agrin  domain, two tandem complement/urchin embryonic growth factor/bone morphogenetic protein  domains, and four tandem low-density lipoprotein receptor class a  domains.
interestingly, matriptase activation does not depend on other active proteases.
instead, several lines of evidence have indicated that matriptase undergoes autoactivation through a mechanism relying on its own catalytic triad and requires its non-catalytic domains as well as the presence of its cognate inhibitor hai- <dig>  <cit> .
although the autoactivation mechanism is not fully understood, one study has showed that matriptase could be activated by acidification, and suggested that matriptase might act as an early response to cellular acidosis  <cit> .
once activated, matriptase has only short time to cleave and activate its substrates since the protease will be quickly inhibited by hai- <dig>
matriptase activates a number of substrates, including g-protein-coupled protease-activated receptor  <dig>  urokinase plasminogen activator and pro-hepatocyte growth factor  <cit> .
recently, it has been demonstrated that matriptase could also activate prekallikren either in vitro or in vivo  <cit> .
matriptase is recognized as a cancer-associated protease since the activation of urokinase plasminogen activator and/or pro-hepatocyte growth factor has been implicated in cancer invasion and metastasis .
in addition, matriptase has been found to be upregulated in various forms of cancers including breast, cervical, ovarian, liver, and prostate cancers.
it has been demonstrated that the level of expression of matriptase correlates with the tumor stage and malignancy of breast, cervical, ovarian and prostate cancers  <cit> .
in some of these cancers, the ratios of the protease relative to its inhibitor hai- <dig> are unbalanced; suggesting that strict regulation of matriptase by hai- <dig> is required to prevent carcinogenesis.
a recent study showed that matriptase orthotopically overexpressed at modest levels in the skin of transgenic mice caused spontaneous squamous cell carcinoma, potentiated chemical carcinogenesis, and supported both ras-dependent and -independent carcinogenesis, whereas the overexpression of hai- <dig> could nullify these oncogenic effects  <cit> .
in addition to its role in cancers, recent studies have suggested that matriptase also has potential implications in a variety of diseases including osteroarthritis, atherosclerosis, and skin disorders like autosomal recessive ichthyosis and hypotrichosis   <cit> .
taken together, matriptase has emerged as an attractive target for the development of anti-metastasis therapy as well as treatment for many other diseases.
sunflower trypsin inhibitor- <dig> , a 14-amino acid cyclic peptide, is originally isolated from sunflower seeds and characterized as the most potent peptidic inhibitor of trypsin   <cit> .
a later study finds that synthetic sfti- <dig> also exhibits very potent matriptase inhibitory activity   <cit> .
to evaluate the structural basis of the high inhibitory effect of sfti- <dig> to matriptase, we have determined the x-ray structure of matriptase in complex with sfti- <dig>  we have also determined the high-resolution structure of matriptase:benzamidine complex for structural comparison.
the crystal structures provide new insights into the molecular basis of matriptase inhibition and this information might facilitate future design of more potent and selective peptide inhibitors using sfti- <dig> as template.
RESULTS
engineering of recombinant matriptase catalytic domain in p. pastoris for structural study
for our structural studies, we constructed a recombinant protease domain of matriptase  with a point mutation n164q , which is referred as β-matriptase-n164q.
the point mutation removes a glycosylation site  and allows the protein to be purified to homogeneity.
another unique feature of the current design of the expression scheme is that the secreted recombinant matriptase protease domain is an active serine protease without the need of being activated.
this is due to the processing of the secreted protein by an endogenous kex <dig> enzyme of p. pastoris that generates the genuine n-terminus of matriptase protease domain and allows the correct folding of the protease into its active form.
we have used such approach to generate a number of active proteases including urokinase-type plasminogen activator  <cit> , tissue-type plasminogen activator and coagulation factor xia , suggesting that our method can be widely adapted for the expression of different active proteases.
structure of β-matriptase-n164q:benzamidine shows benzamidine mobility
in the structure of β-matriptase-n164q complexed with benzamidine, the benzamidine binds to the specificity pocket s <dig> right next to the active site and is sandwiched by the segments of ser190-gln <dig> and trp215-gly <dig> as expected.
this structure is similar to the previously reported structure of the matriptase protease domain in complex with benzamidine   <cit> .
however, there are interesting differences between these two structures.
the phenyl group of the benzamidine in our structure shows a 35° rotation when compared to the 1eax structure.
while the amidino group of the inhibitor juxtaposes the side chain of asp <dig> and forms favorable ionic interaction with its carboxylate group as in the 1eax structure, the bis-amine in our structure is rotated for ~20° and makes an extra hydrogen bond with the backbone carbonyl of ser <dig>  in addition to those formed with the gly <dig> carboxylate and a conserved solvent molecule in 1eax .
considering the high resolutions of both matriptase-benzamidine structures  and the relatively small errors of structures , such differences in benzamidines are significant, and indicates that s <dig> pocket is larger than the molecular size of benzamidine and allows movement of benzamidine inside the s <dig> specificity pocket.
this is consistent with the observed higher temperature factor of benzamidine  comparing to the average temperature factor of protein .
interactions between matriptase and sfti- <dig> at the active site
since sfti- <dig> is a highly potent inhibitor to either matriptase or trypsin with comparable potencies, we want to investigate if the inhibitory mechanisms are the same for the two proteases.
the crystal structure of β-matriptase-n164q in complex with sfti- <dig> was solved at  <dig>  Å .
in this structure, sfti- <dig> forms a bicyclic structure where two antiparallel β-strands are constrained by a disulfide bond between cys <dig> and cys <dig> .
the cyclic peptidic inhibitor docks onto matriptase's p4-p2' specificity sites with good shape and charge complementarity.
this resembles the sfti- <dig> binding to trypsin and other bbi to serine proteases, as well as the reactive site loop of the bovine pancreatic trypsin inhibitor  to matriptase  <cit> .
like the equivalent side chains in the other complexes, lys <dig> from the "reactive loop" of sfti- <dig> extends into the s <dig> pocket of matriptase to form a hydrogen bond directly with ser <dig> and may also interact with asp <dig> at the bottom of the pocket .
the sfti- <dig> scissile bond  is in close proximity to the catalytic residue ser <dig> where the ser <dig> hydroxyl group hydrogen bonds with the main-chain amide of ser <dig> and makes close contacts with the backbone carbonyl and amide of lys <dig> .
the side chain oxygen atom of ser <dig> is also in close contact with the carbonyl carbon of p <dig> residue   at an angle of  <dig> ° , suggesting the protease is in an active conformation capable of initiating bond scission.
furthermore, his <dig> is suitably oriented to interact with ser <dig> to support catalysis .
these observations support that sfti- <dig> functions as a substrate variant of matriptase in a similar manner to other bbis.
thus, the cyclic peptidic inhibitor is likely to inhibit the enzyme through high affinity binding to the active site and through excluding water molecules from the active site to destabilize the reaction-path transition state.
comparison of the conformations of matriptase in the benzamidine- and sfti-1-bound forms reveals that they are nearly identical except the side chain of phe <dig> at the s <dig> subsite, which will be discussed later, and gln <dig> at the s <dig> subsite.
gln <dig> side chain is solvent-exposed in the benzamidine-bound structure and forms the lining of the extended active site groove.
however, it undergoes a major conformational change to "bend inward" to accommodate sfti- <dig> and hydrogen bonds with the backbone carbonyl of thr <dig> of the cyclic inhibitor in a manner similar to that in the bpti-bound matriptase structure.
substrate selectivity of matriptase
previous studies using positional scanning-synthetic combinatorial library and substrate phage display have revealed that matriptase also strongly prefers substrates with small residues including glycine and serine, or phenylalanine at the p <dig> position, in addition to the protease specificity for basic residue at the p <dig> pocket  <cit> .
moreover, the most effective substrate has been found to contain basic residues at the p <dig> or p <dig> sites, but not both simultaneously  <cit> .
indeed, the sequence of sfti- <dig> matches these findings except the presence of a threonine at the p <dig> position.
our structure explains how sfti- <dig> achieves such substrate selectivity.
in the structure, the s <dig> subsite of matriptase is occupied by sfti- <dig> thr <dig> with thr <dig> side chain flanked by his <dig> and phe <dig>  the methyl group of thr <dig> makes ch-π interaction with both flanking side chains and docks snugly in the pocket.
comparing to the benzamidine-bound structure, we observed that the benzyl ring of phe <dig> undergoes a rotation to widen the pocket to better accommodate the p <dig> residue .
similar observation has also been made in the bpti:matriptase complex  <cit> .
this structural explanation for the suitability of threonine at p <dig> position suggests that the s <dig> pocket is not rigid and its size is controlled by the conformation of phe <dig>  a large residue at substrate p <dig> position will inevitably cause steric hindrance with his <dig> and alter its position, and will disrupt the hydrogen bond between asp <dig> and his <dig> and hinder catalysis.
yet, despite its large size, a phenylalanine at p <dig> would make favorable π-π interactions with phe <dig> and his <dig>  and/or cation-π interaction when the histidine is protonated during catalysis, explaining why phenylalanine is also preferred in addition to small residues at this site  <cit> .
comparison with the trypsin:sfti- <dig> complex  reveals that the β-turn consisted of phe <dig>  pro <dig>  asp <dig> and gly <dig> of sfti- <dig> adopts a slightly different conformation upon binding to matriptase  <cit> .
this is to avoid steric hindrance with the matriptase  <dig> loop.
particularly, both side chains of sfti- <dig> phe <dig> and matriptase phe <dig>  undergo large rotational changes to avoid clashing with each other .
in its new conformation, phe <dig> is now stabilized by van der waals interaction with the benzyl rings of phe <dig> and phe <dig>  gly <dig> consequently shifts slightly closer to the active site groove by ~ <dig>  Å, allowing its main-chain carbonyl to make an extra contact with the amide of gly <dig>  interaction between asp <dig> and gln <dig> of trypsin is replaced with intramolecular interaction between asp <dig> and arg <dig> of sfti- <dig>  which stabilizes the conformation of arg <dig> side chain to interact with the s <dig> subsite .
the backbone carbonyls of asp <dig>  phe <dig> and thr <dig> form the s <dig> subsite, which is confined and protected from the solvent by the side chains of phe <dig> and phe <dig>  <cit> .
previous positional scanning and substrate phage display studies performed by takeuchi et al  <cit>  had revealed that the s <dig> subsite of matriptase strongly prefers basic residues.
our structure provides a clear explanation on this selectivity.
firstly, unlike in the trypsin-sfti- <dig> complex, the guanidinium group of arg <dig> is anchored by asp <dig> to form two hydrogen bonds with the carbonyl of phe <dig>  secondly, the rearrangement of the benzyl groups of phe <dig> and phe <dig> results in a solvent-shielding environment that lowers the dielectric constant and thus stabilizes the hydrogen bonding interactions mediated by arg <dig> .
more importantly, the guanidinium group of arg <dig> forms cation-π interactions with both phenylalanines as well as trp <dig> in t-shaped geometries and provides further stabilization  <cit> .
to confirm these interactions, we used the program capture to estimate the stabilizing energies from the cation-π interactions   <cit> .
the interaction between arg <dig> and the s <dig> subsite alone is estimated to contribute ~ <dig> kcal/mol, suggesting that the s <dig> subsite may play a key role in matriptase specificity.
indeed, inhibitory assays with different sfti- <dig> derivatives have shown that the s <dig> subsite strongly prefers arginine  <cit> .
minor modifications of the arg <dig> guanidinium in sfti- <dig> result in significant loss of matriptase inhibition, and replacement of arg <dig> with a phe decreases the inhibitory activity by 900-fold  <cit> .
comparison of sfti- <dig> binding to matriptase and trypsin
matriptase protease domain contains many acidic residues, leading to a lower theoretical pi in comparison with bovine trypsin .
in addition, the electrostatic surface potential of the active site groove of the matriptase protease domain is much more negatively charged than that of trypsin .
thus, the basic sfti- <dig>  would presumably favor matriptase binding than trypsin binding.
however, sfti- <dig> has been shown to inhibit trypsin and matriptase with comparable affinities  <cit> , and we observed here that the binding interface of sfti- <dig> to matriptase is structurally very similar to trypsin.
such apparent discrepancy may be due to the lower mobility of sfti- <dig>  and thus stronger binding, in the current matriptase:sfti- <dig> crystal structure when compared to the trypsin:sfti- <dig> structure .
here the relative mobility is defined by the average temperature factor of sfti- <dig> in the structure of the complexes divided, by the average temperature factor of the rest of protein in order to cancel out the effect of resolution on the temperature factor.
another possible explanation is to take into account the penalty of the loss of configurational entropy of both protein and ligand upon sfti- <dig> binding  <cit> .
for instance, phe <dig> of sfti- <dig> is more constrained in the matriptase complex than in the trypsin complex due to the presence of phe <dig> and phe <dig> that surround it, which might result in a lower torsional degree of freedom and yield an entropy penalty, compensating the gain of enthalpy from the charge complementarity.
implication for future inhibitor design
the current structure provides a template for further improvement of sfti- <dig>  for instance, non-polar residues like ile <dig> and ile <dig> are good candidates for modifications of this bicyclic peptide to improve its binding enthalpy.
ile <dig> is located in a cavity formed by the surface exposed insertion loops  of matriptase and is proximal to the active site.
however, it does not make any direct contacts with residues from the protease.
previous work by li et al.
shows that ile <dig> plays an important role in the selectivity of the inhibitor as its replacement by a more polar and bulky glutamine improves the compound selectivity for matriptase versus thrombin by > <dig> fold  <cit> .
based on the electrostatic surface potential of matriptase calculated from our structure, the modification of sfti- <dig> ile <dig> to a positively charged amino acid might fit the cavity more tightly and provide a favorable enthalpy.
however, increasing flexibility of the inhibitor might result in loss of configurational entropy upon binding, as illustrated by the higher ki of the gln <dig> derivative of sfti- <dig>  <cit> .
therefore, flexible amino acids like lysine and arginine should be avoided for the substitution and small positively charged unnatural amino acids such as diaminopropionic acid or diaminobutyric acid may be used instead.
these small side chains will likely favor the interaction with asp <dig> without disrupting the conformation of the catalytic triad and improves the enthalpy of binding while minimizing the entropy penalty  <cit> .
similarly, ile <dig> lies on top of a groove adjacent to the catalytic cleft of matriptase and its sole direct interaction with matriptase is through weak van der waals interaction with ile <dig>  we believe our suggested strategy for the modification of ile <dig> can also be applied to ile <dig>  together, the combination of modifications at ile <dig> and ile <dig> should improve the inhibitory activity of sfti- <dig> towards matriptase.
CONCLUSIONS
sfti- <dig> is originally isolated and characterized as a potent trypsin inhibitor.
it has also been synthesized by roller's group and shown to exhibit potent inhibitory effect against matriptase.
the same group also investigated the structural basis of the high inhibitory activity of sfti- <dig> using molecular modeling study and obtained information that aids the design and synthesis of new sfti- <dig> analogs.
while modification to stabilize the disulfide bond within the cyclic peptide maintains the compound's inhibitory potency and selectivity of matriptase versus thrombin, replacement of ile <dig> with the more polar glutamine improves selectivity towards matriptase at the expense of weakening its inhibitory activity.
nevertheless, none of the modified inhibitors show improvement in binding affinity to matriptase.
this major drawback can now be overcome by better aid from the structural information of an experimentally obtained structure of matriptase:sfti- <dig> complex.
this work helps to define the structural basis of substrate specificity of matriptase and provides more details in the interactions between the inhibitor and protease.
our structure also reveals the structural difference between the sfti- <dig> bound matriptase and trypsin complexes to allow development of more potent and selective inhibitors for matriptase.

 matriptase is a type ii transmembrane serine protease of the s trypsin-like family. matriptase activity is down-regulated by its physiological inhibitor, hepatocyte growth factor activator inhibitor-. matriptase is expressed in most epithelial cells and plays essential roles in the establishment and maintenance of epithelial integrity. new evidence suggests that matriptase is also expressed on mast cells, peripheral blood monocytes and b cells, implicating matriptase in the physiological and pathologic functions of these cells. knock down studies in mice have shown that the protease is important in postnatal survival, epidermal barrier formation, hair follicle growth and thymichomeostasis. at the same time, genetic studies using zebra fish and mice have indicated that the activity of matriptase is critical for tissue-integrity and function, and must be strictly controlled by hai-. the catalytic domain of matriptase is tethered to the cell surface via its n-terminal signal anchor, linked by a sea urchin sperm protein/enterokinase/agrin domain, two tandem complement/urchin embryonic growth factor/bone morphogenetic protein domains, and four tandem low-density lipoprotein receptor class a domains. interestingly, matriptase activation does not depend on other active proteases. instead, several lines of evidence have indicated that matriptase undergoes autoactivation through a mechanism relying on its own catalytic triad and requires its non-catalytic domains as well as the presence of its cognate inhibitor hai-. although the autoactivation mechanism is not fully understood, one study has showed that matriptase could be activated by acidification, and suggested that matriptase might act as an early response to cellular acidosis. once activated, matriptase has only short time to cleave and activate its substrates since the protease will be quickly inhibited by hai- matriptase activates a number of substrates, including g-protein-coupled protease-activated receptor urokinase plasminogen activator and pro-hepatocyte growth factor. recently, it has been demonstrated that matriptase could also activate prekallikren either in vitro or in vivo. matriptase is recognized as a cancer-associated protease since the activation of urokinase plasminogen activator and/or pro-hepatocyte growth factor has been implicated in cancer invasion and metastasis. in addition, matriptase has been found to be upregulated in various forms of cancers including breast, cervical, ovarian, liver, and prostate cancers. it has been demonstrated that the level of expression of matriptase correlates with the tumor stage and malignancy of breast, cervical, ovarian and prostate cancers. in some of these cancers, the ratios of the protease relative to its inhibitor hai- are unbalanced; suggesting that strict regulation of matriptase by hai- is required to prevent carcinogenesis. a recent study showed that matriptase orthotopically overexpressed at modest levels in the skin of transgenic mice caused spontaneous squamous cell carcinoma, potentiated chemical carcinogenesis, and supported both ras-dependent and -independent carcinogenesis, whereas the overexpression of hai- could nullify these oncogenic effects. in addition to its role in cancers, recent studies have suggested that matriptase also has potential implications in a variety of diseases including osteroarthritis, atherosclerosis, and skin disorders like autosomal recessive ichthyosis and hypotrichosis. taken together, matriptase has emerged as an attractive target for the development of anti-metastasis therapy as well as treatment for many other diseases. sunflower trypsin inhibitor-, a 14-amino acid cyclic peptide, is originally isolated from sunflower seeds and characterized as the most potent peptidic inhibitor of trypsin. a later study finds that synthetic sfti- also exhibits very potent matriptase inhibitory activity. to evaluate the structural basis of the high inhibitory effect of sfti- to matriptase, we have determined the x-ray structure of matriptase in complex with sfti- we have also determined the high-resolution structure of matriptase:benzamidine complex for structural comparison. the crystal structures provide new insights into the molecular basis of matriptase inhibition and this information might facilitate future design of more potent and selective peptide inhibitors using sfti- as template.  engineering of recombinant matriptase catalytic domain in p. pastoris for structural study for our structural studies, we constructed a recombinant protease domain of matriptase with a point mutation n164q, which is referred as β-matriptase-n164q. the point mutation removes a glycosylation site and allows the protein to be purified to homogeneity. another unique feature of the current design of the expression scheme is that the secreted recombinant matriptase protease domain is an active serine protease without the need of being activated. this is due to the processing of the secreted protein by an endogenous kex enzyme of p. pastoris that generates the genuine n-terminus of matriptase protease domain and allows the correct folding of the protease into its active form. we have used such approach to generate a number of active proteases including urokinase-type plasminogen activator, tissue-type plasminogen activator and coagulation factor xia, suggesting that our method can be widely adapted for the expression of different active proteases. structure of β-matriptase-n164q:benzamidine shows benzamidine mobility in the structure of β-matriptase-n164q complexed with benzamidine, the benzamidine binds to the specificity pocket s right next to the active site and is sandwiched by the segments of ser190-gln and trp215-gly as expected. this structure is similar to the previously reported structure of the matriptase protease domain in complex with benzamidine. however, there are interesting differences between these two structures. the phenyl group of the benzamidine in our structure shows a 35° rotation when compared to the 1eax structure. while the amidino group of the inhibitor juxtaposes the side chain of asp and forms favorable ionic interaction with its carboxylate group as in the 1eax structure, the bis-amine in our structure is rotated for ~20° and makes an extra hydrogen bond with the backbone carbonyl of ser in addition to those formed with the gly carboxylate and a conserved solvent molecule in 1eax. considering the high resolutions of both matriptase-benzamidine structures and the relatively small errors of structures, such differences in benzamidines are significant, and indicates that s pocket is larger than the molecular size of benzamidine and allows movement of benzamidine inside the s specificity pocket. this is consistent with the observed higher temperature factor of benzamidine comparing to the average temperature factor of protein. interactions between matriptase and sfti- at the active site since sfti- is a highly potent inhibitor to either matriptase or trypsin with comparable potencies, we want to investigate if the inhibitory mechanisms are the same for the two proteases. the crystal structure of β-matriptase-n164q in complex with sfti- was solved at Å. in this structure, sfti- forms a bicyclic structure where two antiparallel β-strands are constrained by a disulfide bond between cys and cys. the cyclic peptidic inhibitor docks onto matriptase's p4-p2' specificity sites with good shape and charge complementarity. this resembles the sfti- binding to trypsin and other bbi to serine proteases, as well as the reactive site loop of the bovine pancreatic trypsin inhibitor to matriptase. like the equivalent side chains in the other complexes, lys from the "reactive loop" of sfti- extends into the s pocket of matriptase to form a hydrogen bond directly with ser and may also interact with asp at the bottom of the pocket. the sfti- scissile bond is in close proximity to the catalytic residue ser where the ser hydroxyl group hydrogen bonds with the main-chain amide of ser and makes close contacts with the backbone carbonyl and amide of lys. the side chain oxygen atom of ser is also in close contact with the carbonyl carbon of p residue at an angle of °, suggesting the protease is in an active conformation capable of initiating bond scission. furthermore, his is suitably oriented to interact with ser to support catalysis. these observations support that sfti- functions as a substrate variant of matriptase in a similar manner to other bbis. thus, the cyclic peptidic inhibitor is likely to inhibit the enzyme through high affinity binding to the active site and through excluding water molecules from the active site to destabilize the reaction-path transition state. comparison of the conformations of matriptase in the benzamidine- and sfti-1-bound forms reveals that they are nearly identical except the side chain of phe at the s subsite, which will be discussed later, and gln at the s subsite. gln side chain is solvent-exposed in the benzamidine-bound structure and forms the lining of the extended active site groove. however, it undergoes a major conformational change to "bend inward" to accommodate sfti- and hydrogen bonds with the backbone carbonyl of thr of the cyclic inhibitor in a manner similar to that in the bpti-bound matriptase structure. substrate selectivity of matriptase previous studies using positional scanning-synthetic combinatorial library and substrate phage display have revealed that matriptase also strongly prefers substrates with small residues including glycine and serine, or phenylalanine at the p position, in addition to the protease specificity for basic residue at the p pocket. moreover, the most effective substrate has been found to contain basic residues at the p or p sites, but not both simultaneously. indeed, the sequence of sfti- matches these findings except the presence of a threonine at the p position. our structure explains how sfti- achieves such substrate selectivity. in the structure, the s subsite of matriptase is occupied by sfti- thr with thr side chain flanked by his and phe the methyl group of thr makes ch-π interaction with both flanking side chains and docks snugly in the pocket. comparing to the benzamidine-bound structure, we observed that the benzyl ring of phe undergoes a rotation to widen the pocket to better accommodate the p residue. similar observation has also been made in the bpti:matriptase complex. this structural explanation for the suitability of threonine at p position suggests that the s pocket is not rigid and its size is controlled by the conformation of phe a large residue at substrate p position will inevitably cause steric hindrance with his and alter its position, and will disrupt the hydrogen bond between asp and his and hinder catalysis. yet, despite its large size, a phenylalanine at p would make favorable π-π interactions with phe and his and/or cation-π interaction when the histidine is protonated during catalysis, explaining why phenylalanine is also preferred in addition to small residues at this site. comparison with the trypsin:sfti- complex reveals that the β-turn consisted of phe pro asp and gly of sfti- adopts a slightly different conformation upon binding to matriptase. this is to avoid steric hindrance with the matriptase loop. particularly, both side chains of sfti- phe and matriptase phe undergo large rotational changes to avoid clashing with each other. in its new conformation, phe is now stabilized by van der waals interaction with the benzyl rings of phe and phe gly consequently shifts slightly closer to the active site groove by ~ Å, allowing its main-chain carbonyl to make an extra contact with the amide of gly interaction between asp and gln of trypsin is replaced with intramolecular interaction between asp and arg of sfti- which stabilizes the conformation of arg side chain to interact with the s subsite. the backbone carbonyls of asp phe and thr form the s subsite, which is confined and protected from the solvent by the side chains of phe and phe. previous positional scanning and substrate phage display studies performed by takeuchi et al had revealed that the s subsite of matriptase strongly prefers basic residues. our structure provides a clear explanation on this selectivity. firstly, unlike in the trypsin-sfti- complex, the guanidinium group of arg is anchored by asp to form two hydrogen bonds with the carbonyl of phe secondly, the rearrangement of the benzyl groups of phe and phe results in a solvent-shielding environment that lowers the dielectric constant and thus stabilizes the hydrogen bonding interactions mediated by arg. more importantly, the guanidinium group of arg forms cation-π interactions with both phenylalanines as well as trp in t-shaped geometries and provides further stabilization. to confirm these interactions, we used the program capture to estimate the stabilizing energies from the cation-π interactions. the interaction between arg and the s subsite alone is estimated to contribute ~ kcal/mol, suggesting that the s subsite may play a key role in matriptase specificity. indeed, inhibitory assays with different sfti- derivatives have shown that the s subsite strongly prefers arginine. minor modifications of the arg guanidinium in sfti- result in significant loss of matriptase inhibition, and replacement of arg with a phe decreases the inhibitory activity by 900-fold. comparison of sfti- binding to matriptase and trypsin matriptase protease domain contains many acidic residues, leading to a lower theoretical pi in comparison with bovine trypsin. in addition, the electrostatic surface potential of the active site groove of the matriptase protease domain is much more negatively charged than that of trypsin. thus, the basic sfti- would presumably favor matriptase binding than trypsin binding. however, sfti- has been shown to inhibit trypsin and matriptase with comparable affinities, and we observed here that the binding interface of sfti- to matriptase is structurally very similar to trypsin. such apparent discrepancy may be due to the lower mobility of sfti- and thus stronger binding, in the current matriptase:sfti- crystal structure when compared to the trypsin:sfti- structure. here the relative mobility is defined by the average temperature factor of sfti- in the structure of the complexes divided, by the average temperature factor of the rest of protein in order to cancel out the effect of resolution on the temperature factor. another possible explanation is to take into account the penalty of the loss of configurational entropy of both protein and ligand upon sfti- binding. for instance, phe of sfti- is more constrained in the matriptase complex than in the trypsin complex due to the presence of phe and phe that surround it, which might result in a lower torsional degree of freedom and yield an entropy penalty, compensating the gain of enthalpy from the charge complementarity. implication for future inhibitor design the current structure provides a template for further improvement of sfti- for instance, non-polar residues like ile and ile are good candidates for modifications of this bicyclic peptide to improve its binding enthalpy. ile is located in a cavity formed by the surface exposed insertion loops of matriptase and is proximal to the active site. however, it does not make any direct contacts with residues from the protease. previous work by li et al. shows that ile plays an important role in the selectivity of the inhibitor as its replacement by a more polar and bulky glutamine improves the compound selectivity for matriptase versus thrombin by > fold. based on the electrostatic surface potential of matriptase calculated from our structure, the modification of sfti- ile to a positively charged amino acid might fit the cavity more tightly and provide a favorable enthalpy. however, increasing flexibility of the inhibitor might result in loss of configurational entropy upon binding, as illustrated by the higher ki of the gln derivative of sfti-. therefore, flexible amino acids like lysine and arginine should be avoided for the substitution and small positively charged unnatural amino acids such as diaminopropionic acid or diaminobutyric acid may be used instead. these small side chains will likely favor the interaction with asp without disrupting the conformation of the catalytic triad and improves the enthalpy of binding while minimizing the entropy penalty. similarly, ile lies on top of a groove adjacent to the catalytic cleft of matriptase and its sole direct interaction with matriptase is through weak van der waals interaction with ile we believe our suggested strategy for the modification of ile can also be applied to ile together, the combination of modifications at ile and ile should improve the inhibitory activity of sfti- towards matriptase.  sfti- is originally isolated and characterized as a potent trypsin inhibitor. it has also been synthesized by roller's group and shown to exhibit potent inhibitory effect against matriptase. the same group also investigated the structural basis of the high inhibitory activity of sfti- using molecular modeling study and obtained information that aids the design and synthesis of new sfti- analogs. while modification to stabilize the disulfide bond within the cyclic peptide maintains the compound's inhibitory potency and selectivity of matriptase versus thrombin, replacement of ile with the more polar glutamine improves selectivity towards matriptase at the expense of weakening its inhibitory activity. nevertheless, none of the modified inhibitors show improvement in binding affinity to matriptase. this major drawback can now be overcome by better aid from the structural information of an experimentally obtained structure of matriptase:sfti- complex. this work helps to define the structural basis of substrate specificity of matriptase and provides more details in the interactions between the inhibitor and protease. our structure also reveals the structural difference between the sfti- bound matriptase and trypsin complexes to allow development of more potent and selective inhibitors for matriptase. 
we have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:sfti- complex at Å as well as the protease:benzamidine complex at Å. these structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase s substrate specificity pocket is larger enough to allow movement of benzamidine inside the s pocket. matriptase is a type ii transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. this work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. sunflower trypsin inhibitor-, a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase.
 matriptase is a type ii transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. matriptase has been implicated in the degradation of certain extracellular matrix components as well as the activation of various cellular proteins and proteases, including hepatocyte growth factor and urokinase. sunflower trypsin inhibitor-, a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase.  we have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:sfti- complex at Å as well as the protease:benzamidine complex at Å. these structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase s substrate specificity pocket is larger enough to allow movement of benzamidine inside the s pocket. our study also reveals that sfti- binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins.  this work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. the complex structure also provides a structural template for designing new sfti- derivatives with better potency and selectivity against matriptase and other proteases. 