title: Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.
abstract: Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain).
authors.affiliation: Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Zebin
authors.initials: Z
authors.lastname: Hong
authors.affiliation: Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Laura
authors.initials: L
authors.lastname: De Meulemeester
authors.affiliation: Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Annemarie
authors.initials: A
authors.lastname: Jacobi
authors.affiliation: Department of Chemistry and iNANO Interdisciplinary Nanoscience Center, Aarhus University, 8000 Aarhus C, Denmark.
authors.forename: Jan Skov
authors.initials: JS
authors.lastname: Pedersen
authors.affiliation: Norwegian Center of Molecular Medicine (NCMM), University of Oslo, NO-0316 Oslo, Norway.
authors.forename: J Preben
authors.initials: JP
authors.lastname: Morth
authors.affiliation: Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.affiliation: Department of Molecular Biology and Genetics, Danish-Chinese Centre for Proteases and Cancer, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark,. Electronic address: jkj@mbg.au.dk.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007690:Polycystic Kidney Diseases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D008562:Membrane Glycoproteins; D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C504591:Spint1 protein, mouse
keywords: cell surface protein; inhibition mechanism; multidomain protein; protease inhibitor; protein-protein interaction; tertiary structure