title: Structural basis of inhibition revealed by a 1:2 complex of the two-headed tomato inhibitor-II and subtilisin Carlsberg.
abstract: Multidomain proteinase inhibitors play critical roles in the defense of plants against predation by a wide range of pests. Despite a wealth of structural information on proteinase-single domain inhibitor interactions, the structural basis of inhibition by multidomain proteinase inhibitors remains poorly understood. Here we report the 2.5-A resolution crystal structure of the two-headed tomato inhibitor-II (TI-II) in complex with two molecules of subtilisin Carlsberg; it reveals how a multidomain inhibitor from the Potato II family of proteinase inhibitors can bind to and simultaneously inhibit two enzyme molecules within a single ternary complex. The N terminus of TI-II initiates the folding of Domain I (Lys-1 to Cys-15 and Pro-84 to Met-123) and then completes Domain II (Ile-26 to Pro-74) before coming back to complete the rest of Domain I (Pro-84 to Met-123). The two domains of TI-II adopt a similar fold and are arranged in an extended configuration that presents two reactive site loops at the opposite ends of the inhibitor molecule. Each subtilisin molecule interacts with a reactive site loop of TI-II through the standard, canonical binding mode. Remarkably, a significant distortion of the active site of subtilisin is induced by the presence of phenylalanine in the P1 position of reactive site loop II of TI-II. The structure of the TI-II.(subtilisin)2 complex provides a molecular framework for understanding how multiple inhibitory domains in a single Potato II type proteinase inhibitor molecule from the Potato II family act to inhibit proteolytic enzymes.
authors.affiliation: Canadian Institutes for Health Research Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
authors.forename: Isabelle H
authors.initials: IH
authors.lastname: Barrette-Ng
authors.forename: Kenneth K-S
authors.initials: KK
authors.lastname: Ng
authors.forename: Maia M
authors.initials: MM
authors.lastname: Cherney
authors.forename: Gregory
authors.initials: G
authors.lastname: Pearce
authors.forename: Clarence A
authors.initials: CA
authors.lastname: Ryan
authors.forename: Michael N G
authors.initials: MN
authors.lastname: James
mesh_terms: D000595:Amino Acid Sequence; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D010940:Plant Proteins; D011480:Protease Inhibitors; D013381:Subtilisins