title: Structural Basis for Action of the External Chaperone for a Propeptide-deficient Serine Protease from Aeromonas sobria.
abstract: Subtilisin-like proteases are broadly expressed in organisms ranging from bacteria to mammals. During maturation of these enzymes, N-terminal propeptides function as intramolecular chaperones, assisting the folding of their catalytic domains. However, we have identified an exceptional case, the serine protease from Aeromonas sobria (ASP), that lacks a propeptide. Instead, ORF2, a protein encoded just downstream of asp, appears essential for proper ASP folding. The mechanism by which ORF2 functions remains an open question, because it shares no sequence homology with any known intramolecular propeptide or other protein. Here we report the crystal structure of the ORF2-ASP complex and the solution structure of free ORF2. ORF2 consists of three regions: an N-terminal extension, a central body, and a C-terminal tail. Together, the structure of the central body and the C-terminal tail is similar to that of the intramolecular propeptide. The N-terminal extension, which is not seen in other subtilisin-like enzymes, is intrinsically disordered but forms some degree of secondary structure upon binding ASP. We also show that C-terminal (ΔC1 and ΔC5) or N-terminal (ΔN43 and ΔN64) deletion eliminates the ability of ORF2 to function as a chaperone. Characterization of the maturation of ASP with ORF2 showed that folding occurs in the periplasmic space and is followed by translocation into extracellular space and dissociation from ORF2, generating active ASP. Finally, a PSI-BLAST search revealed that operons encoding subtilases and their external chaperones are widely distributed among Gram-negative bacteria, suggesting that ASP and its homologs form a novel family of subtilases having an external chaperone.
authors.affiliation: From the Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima 737-0112, Japan.
authors.forename: Hidetomo
authors.initials: H
authors.lastname: Kobayashi
authors.affiliation: the Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: the Department of Chemistry, College of Science and Technology, Meisei University, Hodokubo, Hino, Tokyo 191-0042, Japan, and.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Tashiro
authors.affiliation: the Department of Pharmacogenetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
authors.forename: Keinosuke
authors.initials: K
authors.lastname: Okamoto
authors.affiliation: From the Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Kure, Hiroshima 737-0112, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Yamanaka
authors.affiliation: the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.affiliation: the Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-Motoyama, Kyoto 603-8555, Japan, tsuge@cc.kyoto-su.ac.jp.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
mesh_terms: D000333:Aeromonas / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D017434:Protein Structure, Tertiary; D057057:Serine Proteases; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D057057:Serine Proteases
keywords: Chaperone; External Chaperone; Intrinsically Disordered Protein; Protein-Protein Interaction; Serine Protease; Steric Chaperone; X-ray Crystallography