Structure of catalytic domain of Matriptase in complex with Sunflower trypsin inhibitor-1
© Yuan et al; licensee BioMed Central Ltd. 2011
Received: 7 February 2011
Accepted: 22 June 2011
Published: 22 June 2011
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-1 (SFTI-1), 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-1 complex at 2.0 Å as well as the protease:benzamidine complex at 1.2 Å. These structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase S1 substrate specificity pocket is larger enough to allow movement of benzamidine inside the S1 pocket. Our study also reveals that SFTI-1 binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins (5.6 vs. 8.2).
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-1 derivatives with better potency and selectivity against matriptase and other proteases.
Matriptase is a type II transmembrane serine protease of the S1 trypsin-like family. Matriptase activity is down-regulated by its physiological inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1) [1–3]. 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 [4–6]. 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-1 [8–11].
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 (SEA) domain, two tandem complement/urchin embryonic growth factor/bone morphogenetic protein (CUB) domains, and four tandem low-density lipoprotein receptor class A (LDLRA) 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-1 [12, 13]. 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-1.
Matriptase activates a number of substrates, including G-protein-coupled protease-activated receptor 2, urokinase plasminogen activator and pro-hepatocyte growth factor [15, 16]. 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 (reviewed in ). 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 [19–21]. In some of these cancers, the ratios of the protease relative to its inhibitor HAI-1 are unbalanced; suggesting that strict regulation of matriptase by HAI-1 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-1 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 (ARIH) [4, 23–26]. 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-1 (SFTI-1), a 14-amino acid cyclic peptide, is originally isolated from sunflower seeds and characterized as the most potent peptidic inhibitor of trypsin (Ki = 0.1 nM and 1 nM from two independent studies) [27, 28]. A later study finds that synthetic SFTI-1 also exhibits very potent matriptase inhibitory activity (Ki = 0.92 nM) . To evaluate the structural basis of the high inhibitory effect of SFTI-1 to matriptase, we have determined the X-ray structure of matriptase in complex with SFTI-1. 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-1 as template.
Results and Discussion
Engineering of recombinant matriptase catalytic domain in P. pastoris for structural study
For our structural studies, we constructed a recombinant protease domain of matriptase (residue 615 to 854 of the EXPASY entry Q9Y5Y6) with a point mutation N164Q (chymotrypsin numbering will be used throughout the paper starting from here), which is referred as β-matriptase-N164Q. The point mutation removes a glycosylation site (N164) 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 kex2 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 (to be published), suggesting that our method can be widely adapted for the expression of different active proteases.
Structure of β-matriptase-N164Q:benzamidine shows benzamidine mobility
Interactions between matriptase and SFTI-1 at the active site
Hydrogen bonds between matriptase (chymotrypsin numbering) and SFTI-1
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 Phe99 at the S2 subsite, which will be discussed later, and Gln192 at the S3 subsite. Gln192 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-1 and hydrogen bonds with the backbone carbonyl of Thr4 of the cyclic inhibitor in a manner similar to that in the BPTI-bound matriptase structure.
Substrate selectivity of matriptase
Calculated binding energies of cation-π interactions between matriptase and SFTI-1 
SFTI-1 side chain
Matriptase aromatic side chain
E(van der Waals) (kcal/mol)
Comparison of SFTI-1 binding to matriptase and trypsin
Implication for future inhibitor design
The current structure provides a template for further improvement of SFTI-1. For instance, non-polar residues like Ile7 and Ile10 are good candidates for modifications of this bicyclic peptide to improve its binding enthalpy. Ile10 is located in a cavity formed by the surface exposed insertion loops (loops 66 and 99) 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 Ile10 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 >1073 fold . Based on the electrostatic surface potential of matriptase calculated from our structure, the modification of SFTI-1 Ile10 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 Gln10 derivative of SFTI-1 . 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 Asp96 without disrupting the conformation of the catalytic triad and improves the enthalpy of binding while minimizing the entropy penalty . Similarly, Ile7 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 Ile41. We believe our suggested strategy for the modification of Ile10 can also be applied to Ile7. Together, the combination of modifications at Ile7 and Ile10 should improve the inhibitory activity of SFTI-1 towards matriptase.
SFTI-1 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-1 using molecular modeling study and obtained information that aids the design and synthesis of new SFTI-1 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 Ile10 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-1 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-1 bound matriptase and trypsin complexes to allow development of more potent and selective inhibitors for matriptase.
Recombinant protein expression and Purification
The matriptase N164Q (chymotrypsin numbering) catalytic domain mutant was first generated by site-directed mutagenesis using cDNA encoding the entire protease domain of human matriptase as template. The cDNA was then amplified by PCR using primers containing XhoI and SalI restriction sites. The purified PCR products were digested with XhoI and SalI and subcloned into the XhoI-SalI sites of the Pichia pastoris (P. pastoris) expression vector pPICZαA (Invitrogen). Plasmid DNAs were linearized with the restriction enzyme SacI prior to transformation into P. pastoris strain X-33. Recombinant matriptases were expressed in P. pastoris according to the manufacturer's recommendations. P. pastoris expression medium was concentrated 10-20 fold using a Millipore concentrator (8000 Da MWCO membrane) and pH was adjusted to 7.4. The concentrated medium was applied onto a benzamidine column (GE Healthcare) equilibrated with 50 mM Tris, 0.5 M NaCl, pH7.4, and eluted with 100 mM glycine, pH 3.0. The elution fractions were neutralized with 1 M Tris pH 9 immediately. Fractions containing matriptase activity were pooled and concentrated, and passed through MonoQ column (Amersham Biosciences, Inc.) pre-equilibrated with 40 mM Tris. The protein was eluted in a buffer containing 40 mM Tris, pH7.4 with a 0-0.4 M NaCl gradient. Fractions containing protein were pooled and concentrated to 5 mg/ml. Aliquots of the purified protein were frozen at 193 K for crystallization experiments.
Crystallization of β-matriptase-N164Q complex with its inhibitors
For protein crystallization, β-matriptase-N164Q:benzamidine complexes were mixed at 1:10 ratio, and crystallized by the hanging-drop vapor diffusion method with a precipitant solution of 0.1 M Tris, pH 8.0, 1.5 M ammonium sulfate, 3% ethanol. SFTI-1 was synthesized by solid state synthesis as previously reported . The complex of β-matriptase-N164Q with SFTI-1 was crystallized with a precipitant condition of 22% polyethylene glycol 8000, 0.1 M Tris pH 8 and 20 mM CaCl2.
Data collection, structure solution and refinement
Statistics of X-ray diffraction data collection and structure refinement
Cell parameters (Å)
66.9, 141.7, 52.0
75.9, 75.9, 94.1
Bond length (Å)
Bond angles (°)
Mean B factors (Å2)
Ramachandran plot, % residues in regions:
PDB ID Code
bovine pancreatic trypsin inhibitor
hepatocyte growth factor activator inhibitor-1
Protein Data Bank
root mean square deviation
Sunflower trypsin inhibitor-1.
The work was supported by Natural Science Foundation of China (30770429, 30811130467) and Fujian Province (2009J05091).
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