title: A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.
abstract: A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark, tobiaskh@mbg.au.dk.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kromann-Hansen
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Emil
authors.initials: E
authors.lastname: Oldenburg
authors.affiliation: the School of Life Sciences, Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region, China.
authors.forename: Kristen Wing Yu
authors.initials: KW
authors.lastname: Yung
authors.affiliation: the Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussels, 1050 Brussels, Belgium, Nanobody Service Facility, Flanders Institute for Biotechnology, 1050 Brussels, Belgium.
authors.forename: Gholamreza H
authors.initials: GH
authors.lastname: Ghassabeh
authors.affiliation: the Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussels, 1050 Brussels, Belgium.
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.affiliation: the Laboratory for Therapeutic and Diagnostic Antibodies, Department of Pharmaceutical and Pharmacological Sciences, Katholieke Universiteit, 3000 Leuven, Belgium, and.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.affiliation: the State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Science, Fuzhou, Fujian 350002, China.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.affiliation: From the Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.affiliation: the School of Life Sciences, Chinese University of Hong Kong, Shatin, Hong Kong Special Administrative Region, China.
authors.forename: Jacky Chi Ki
authors.initials: JC
authors.lastname: Ngo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D020134:Catalytic Domain; D022801:Complementarity Determining Regions; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D057057:Serine Proteases / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology
chemical_list: D022801:Complementarity Determining Regions; D015842:Serine Proteinase Inhibitors; D061905:Single-Domain Antibodies; D057057:Serine Proteases; D014568:Urokinase-Type Plasminogen Activator
keywords: biophysics; biotechnology; serine protease; single-domain antibody (sdAb, nanobody); x-ray crystallography