Abstract
The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 A and bovine pancreatic trypsin inhibitor at 2.9 A. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.
Highlights
The activity of proteolytic enzymes is required at multiple stages during the growth, invasion, and progression of human tumors
The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MTSP1, and suggests determinants for the substrate specificity
Because the matriptase sequence reported by Lin turned out to be part of the translated MT-SP1 cDNA sequence, matriptase is likely to be a form of MT-SP1 produced by ectodomain shedding [7]
Summary
Cloning and Purification—The human prostate adenocarcinoma cell line, PC-3, was purchased from ATCC (CRL-1435). The 5Ј end of the MT-SP1 proteinase domain was obtained by a PCR amplification reaction using two MTSP1-specific primers, 5Ј-TACCTCTCCTACGACTCC-3Ј for the sense primer and 5Ј-GAGGTTCTCGCAGGTGGTCTGGTTG-3Ј for the antisense primer Crystallization, Structure Determination, and Crystallographic Refinement—Plate-like crystals of the Bz-MT-SP1 complex were grown from 0.1 M Tris-HCl, pH 8.0, 1.5 M ammonium sulfate, 3% ethanol at 18 °C using the hanging drop vapor diffusion technique. These crystals belong to the orthorhombic space group C222, diffract x-rays to beyond 1.3-Å resolution, and have one molecule in the asymmetric unit.
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