Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Feld, Geoffrey K.; El-Etr, Sahar; Corzett, Michele H.; Hunter, Mark S.; Belhocine, Kamila; Monack, Denise M.; Frank, Matthias; Segelke, Brent W.; Rasley, Amy

doi:10.1074/jbc.m114.599381

Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Journal Article · Sun Jan 03 23:00:00 EST 2021 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.m114.599381· OSTI ID:1840848

Feld, Geoffrey K. ^[1]; El-Etr, Sahar ^[1]; Corzett, Michele H. ^[1]; Hunter, Mark S. ^[1]; Belhocine, Kamila ^[2]; Monack, Denise M. ^[3]; Frank, Matthias ^[1]; Segelke, Brent W. ^[1]; Rasley, Amy ^[1]

Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Stanford Univ. School of Medicine, CA (United States); Theranos, Inc., Palo Alto, CA (United States)
Stanford Univ. School of Medicine, CA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States)

Sponsoring Organization:: USDOE National Nuclear Security Administration (NNSA)

Grant/Contract Number:: AC52-07NA27344

OSTI ID:: 1840848

Alternate ID(s):: OSTI ID: 1164567

Report Number(s):: LLNL-JRNL--656280; 777061

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 44 Vol. 289; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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