title: Structure and function of REP34 implicates carboxypeptidase activity in Francisella tularensis host cell invasion.
abstract: Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.
authors.affiliation: Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550.
authors.forename: Geoffrey K
authors.initials: GK
authors.lastname: Feld
authors.affiliation: Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550.
authors.forename: Sahar
authors.initials: S
authors.lastname: El-Etr
authors.affiliation: Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550.
authors.forename: Michele H
authors.initials: MH
authors.lastname: Corzett
authors.affiliation: Physics Divisions, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 and.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hunter
authors.affiliation: Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Kamila
authors.initials: K
authors.lastname: Belhocine
authors.affiliation: Stanford University School of Medicine, Stanford, California 94305.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Monack
authors.affiliation: Physics Divisions, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 and.
authors.forename: Matthias
authors.initials: M
authors.lastname: Frank
authors.affiliation: Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550.
authors.forename: Brent W
authors.initials: BW
authors.lastname: Segelke
authors.affiliation: Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550. Electronic address: rasley2@llnl.gov.
authors.forename: Amy
authors.initials: A
authors.lastname: Rasley
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000502:physiology; D002268:Carboxypeptidases / Q000737:chemistry* / Q000502:physiology; D020134:Catalytic Domain; D002460:Cell Line; D005604:Francisella tularensis / Q000502:physiology*; D054884:Host-Pathogen Interactions; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009000:Monocytes / Q000382:microbiology; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D002268:Carboxypeptidases
keywords: Carboxypeptidase; Enzyme Structure; Francisella tularensis; Peptidase; Virulence Factor; X-ray Crystallography