Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion |
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Authors: | Geoffrey K Feld Sahar El-Etr Michele H Corzett Mark S Hunter Kamila Belhocine Denise M Monack Matthias Frank Brent W Segelke Amy Rasley |
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Institution: | From the ‡Biosciences and Biotechnology and ;§Physics Divisions, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 and ;the ¶Stanford University School of Medicine, Stanford, California 94305 |
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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. |
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Keywords: | Carboxypeptidase Enzyme Structure Peptidase Virulence Factor X-ray Crystallography Francisella tularensis |
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