Abstract
BackgroundMatriptase 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.ResultsWe 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).ConclusionsThis 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.
Highlights
Matriptase is a type II transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells
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 b-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
Summary
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. Matriptase is a type II transmembrane serine protease of the S1 trypsin-like family. Matriptase activity is downregulated by its physiological inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1) [1,2,3]. Knock down studies in mice have shown that the protease is important in postnatal survival, epidermal barrier formation, hair follicle growth and thymichomeostasis [7]. Genetic studies using zebra fish and mice have indicated that the activity of
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