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

  • Geoffrey K. Feld
    Affiliations
    Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550
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  • Sahar El-Etr
    Affiliations
    Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550
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  • Michele H. Corzett
    Affiliations
    Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550
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  • Mark S. Hunter
    Affiliations
    Physics Divisions, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 and
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  • Kamila Belhocine
    Footnotes
    Affiliations
    Stanford University School of Medicine, Stanford, California 94305
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  • Denise M. Monack
    Affiliations
    Stanford University School of Medicine, Stanford, California 94305
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  • Matthias Frank
    Affiliations
    Physics Divisions, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550 and
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  • Brent W. Segelke
    Affiliations
    Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550
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  • Amy Rasley
    Correspondence
    To whom correspondence should be addressed: Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, 7000 East Ave., L452, Livermore, CA 94550. Tel.: 925-423-1284
    Affiliations
    Biosciences and Biotechnology and Lawrence Livermore National Laboratory, Livermore, California 94550
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health (NIH), NIAID, Grant AI65359 (to A. R.). This research was also supported by Lawrence Livermore National Laboratory (LLNL) Laboratory-directed Research and Development (LDRD) Project 06-ERD-057 (to A. R.) and LDRD Project 012-ERD-031 (to M. F.). Work was performed under the auspices of the United States Department of Energy by Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. Use of the Stanford Synchrotron Radiation Laboratory (SSRL), SLAC National Accelerator Laboratory, was supported by the United States Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, NIGMS (including Grant P41GM103393). GM/CA at the Advanced Photon Source (APS) has been funded in whole or in part with Federal funds from NIH, NCI, Grant Y1-CO-1020 and NIH, NIGMS, Grant Y1-GM-1104. The Northeastern Collaborative Access Team is supported by NIH, NCRR, Grant 5P41RR015301-10 and NIH, NIGMS, Grant P41 GM103403. Use of the APS is supported by the DOE under Contract DE-AC02-06CH11357.The atomic coordinates and structure factors (code 4OKO) have been deposited in the Protein Data Bank (http://wwpdb.org/).
    4 Secondary structure elements are numbered according to funnelin convention.
    1 Present address: Theranos, Inc., Palo Alto, CA 94304.
    3 The abbreviations used are: LVSlive vaccine strainREPrapid encystment phenotyperREP34recombinant REP34CPACPB, and CPT, carboxypeptidase A, B, and T, respectivelyNOVF. tularensis subspecies novicida U112PDBProtein Data BankSADsingle wavelength anomalous diffractionGEMSA2-guanidinoethylmercaptosuccinic acid.
      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.

      Introduction

      Francisella tularensis is a Gram-negative bacteria and the cause of the zoonotic disease tularemia, a debilitating, acute disease with high mortality rates if untreated (
      • Oyston P.C.
      Francisella tularensis: unravelling the secrets of an intracellular pathogen.
      ,
      • Clemens D.L.
      • Horwitz M.A.
      Uptake and intracellular fate of Francisella tularensis in human macrophages.
      ). Also known as rabbit fever, due to its historical propensity to infect hunters and trappers, tularemia has recently appeared in diverse geographic regions, where it was either previously unseen (
      • Eliasson H.
      • Back E.
      Tularaemia in an emergent area in Sweden: an analysis of 234 cases in five years.
      ,
      • Gürcan S.
      • Eskiocak M.
      • Varol G.
      • Uzun C.
      • Tatman-Otkun M.
      • Sakru N.
      • Karadenizli A.
      • Karagöl C.
      • Otkun M.
      Tularemia re-emerging in European part of Turkey after 60 years.
      ) or reemerged after more than 20 years dormancy (
      • Feldman K.A.
      • Enscore R.E.
      • Lathrop S.L.
      • Matyas B.T.
      • McGuill M.
      • Schriefer M.E.
      • Stiles-Enos D.
      • Dennis D.T.
      • Petersen L.R.
      • Hayes E.B.
      An outbreak of primary pneumonic tularemia on Martha's Vineyard.
      ). Subspecies of F. tularensis are facultative intracellular pathogens that exhibit remarkably broad host ranges, capable of infecting more than 200 diverse hosts and vectors (
      • Ellis J.
      • Oyston P.C.
      • Green M.
      • Titball R.W.
      Tularemia.
      ,
      • Nigrovic L.E.
      • Wingerter S.L.
      Tularemia.
      • Petersen J.M.
      • Mead P.S.
      • Schriefer M.E.
      Francisella tularensis: an arthropod-borne pathogen.
      ). The organism's geographic and infectious diversity, combined with increased awareness of its potential as a biothreat weapon (
      • Oyston P.C.
      • Sjostedt A.
      • Titball R.W.
      Tularaemia: bioterrorism defence renews interest in Francisella tularensis.
      ) and the absence of an approved vaccine for prophylaxis against tularemia (
      • Oyston P.C.
      Francisella tularensis: unravelling the secrets of an intracellular pathogen.
      ) contribute to classification of F. tularensis as a Tier 1 priority pathogen by the Centers for Disease Control.
      Berdal et al. (
      • Berdal B.P.
      • Mehl R.
      • Meidell N.K.
      • Lorentzen-Styr A.M.
      • Scheel O.
      Field investigations of tularemia in Norway.
      ) first reported the association of F. tularensis with the free-living amoeba, Acanthamoeba castellanii, and proposed that these organisms may act as a potential environmental reservoir for the pathogen in Norway. Using the F. tularensis subspecies holarctica live vaccine strain (LVS),
      The abbreviations used are: LVS
      live vaccine strain
      REP
      rapid encystment phenotype
      rREP34
      recombinant REP34
      CPA
      CPB, and CPT, carboxypeptidase A, B, and T, respectively
      NOV
      F. tularensis subspecies novicida U112
      PDB
      Protein Data Bank
      SAD
      single wavelength anomalous diffraction
      GEMSA
      2-guanidinoethylmercaptosuccinic acid.
      Abd et al. (
      • Abd H.
      • Johansson T.
      • Golovliov I.
      • Sandström G.
      • Forsman M.
      Survival and growth of Francisella tularensis in Acanthamoeba castellanii.
      ) confirmed the ability of F. tularensis to infiltrate A. castellanii cells, further implicating these protozoa in the pathogen's environmental persistence. It is well documented that A. castellanii can serve as a reservoir for a number of pathogenic microorganisms (
      • Winiecka-Krusnell J.
      • Dellacasa-Lindberg I.
      • Dubey J.P.
      • Barragan A.
      Toxoplasma gondii: uptake and survival of oocysts in free-living amoebae.
      • Steinert M.
      • Birkness K.
      • White E.
      • Fields B.
      • Quinn F.
      Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls.
      ,
      • Salah I.B.
      • Ghigo E.
      • Drancourt M.
      Free-living amoebae, a training field for macrophage resistance of mycobacteria.
      ,
      • Lauriano C.M.
      • Barker J.R.
      • Yoon S.S.
      • Nano F.E.
      • Arulanandam B.P.
      • Hassett D.J.
      • Klose K.E.
      MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival.
      ,
      • Winiecka-Krusnell J.
      • Linder E.
      Bacterial infections of free-living amoebae.
      • Miyake M.
      • Watanabe T.
      • Koike H.
      • Molmeret M.
      • Imai Y.
      • Abu Kwaik Y.
      Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages.
      ) and enhance the virulence of these organisms in murine models of infection (
      • Cirillo J.D.
      • Falkow S.
      • Tompkins L.S.
      • Bermudez L.E.
      Interaction of Mycobacterium avium with environmental amoebae enhances virulence.
      ,
      • Brieland J.
      • McClain M.
      • Heath L.
      • Chrisp C.
      • Huffnagle G.
      • LeGendre M.
      • Hurley M.
      • Fantone J.
      • Engleberg C.
      Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires' disease.
      ). Selective pressure for survival from protozoa predation has been suggested to represent a driving force in the evolution of pathogenic bacteria that persist in the environment (
      • Miyake M.
      • Watanabe T.
      • Koike H.
      • Molmeret M.
      • Imai Y.
      • Abu Kwaik Y.
      Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages.
      ). Given that macrophages in higher organisms possess similar physiology and phagocytic ability as amoebae, protozoa may serve as “training grounds” for intracellular pathogens by priming the bacteria for survival in phagocytic cells (
      • Molmeret M.
      • Horn M.
      • Wagner M.
      • Santic M.
      • Abu Kwaik Y.
      Amoebae as training grounds for intracellular bacterial pathogens.
      ). Because F. tularensis preferentially infects and replicates within host macrophages, probing the Francisella-amoeba interaction may provide insight into pathogenesis as well as environmental persistence.
      We recently demonstrated that fully virulent F. tularensis isolates infect A. castellanii by inducing the amoebae to rapidly encyst (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ). The study identified seven putatively secreted F. tularensis proteins that may be responsible for this rapid encystment phenotype (REP) in amoeba, a phenomenon required for long term survival of F. tularensis in these hosts. Interestingly, two of the genes encoding these REP proteins are deleted in LVS, consistent with the inability of LVS to induce amoeba encystment (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ) and with its attenuated infectivity toward macrophages (
      • Oyston P.C.
      Francisella tularensis: unravelling the secrets of an intracellular pathogen.
      ). Here, we focus on the gene product of FTN_0149 from F. tularensis subspecies novicida U112 (NOV), a REP protein with a molecular mass of 34 kDa (REP34).
      The amino acid sequence of REP34 indicates weak sequence homology to the M14 family of metallocarboxypeptidases (
      • Rawlings N.D.
      • Barrett A.J.
      Evolutionary families of peptidases.
      ) and even less homology toward metallocarboxypeptidases for which structures are available in the PDB. M14 metallocarboxypeptidases are a diverse and important class of peptidases that catalyze the removal of the C-terminal residue from polypeptides by means of a coordinated Zn2+ cofactor. Carboxypeptidase A (CPA)-like enzymes catalyze the removal of a hydrophobic residue, whereas carboxypeptidase B (CPB)-like peptidases remove basic C-terminal residues. The bacterial homologue carboxypeptidase T (CPT) can catalyze both CPA- and CPB-like reactions (
      • Stepanov V.M.
      Carboxypeptidase T.
      ). Regardless of the substrate, the Zn2+-dependent carboxypeptidases share a common α/β hydrolase-like fold and a conserved active site. The active site consists of a distorted tetrahedral Zn2+ coordination site (usually a pair of histidines, a glutamate, and a coordinated water molecule) surrounded by catalytic residues that stabilize intermediates during hydrolysis. Substrate molecules access the active site via a cleft on the enzyme surface, giving the enzyme a distinct funnel shape. Thus, members of this class of peptidases are referred to as funnelins (
      • Gomis-Rüth F.X.
      Structure and mechanism of metallocarboxypeptidases.
      ).
      Metallocarboxypeptidases have been previously implicated in virulence. Insertional inactivation of CPG70, a secreted metallocarboxypeptidase with CPB-like activity from Porphyromonas gingivalis, resulted in an avirulent bacterial phenotype in a murine model (
      • Chen Y.Y.
      • Cross K.J.
      • Paolini R.A.
      • Fielding J.E.
      • Slakeski N.
      • Reynolds E.C.
      CPG70 is a novel basic metallocarboxypeptidase with C-terminal polycystic kidney disease domains from Porphyromonas gingivalis.
      ). The dermatophyte Trichophyton rubrum secretes the CPA-like prepeptidase TruMcpA, and the mature peptidase contributes to the virulence of these fungi, along with a host of additional serine carboxypeptidases (
      • Zaugg C.
      • Jousson O.
      • Léchenne B.
      • Staib P.
      • Monod M.
      Trichophyton rubrum secreted and membrane-associated carboxypeptidases.
      ). Although the host targets of these various peptidases are unknown, it is believed that they work in concert with other endo- and aminopeptidases to promote infection.
      In this study, we characterize the cellular and molecular function of REP34 to better understand the biological role of REP in F. tularensis infection and environmental persistence. The comprehensive MEROPS peptidase database (
      • Rawlings N.D.
      • Barrett A.J.
      • Bateman A.
      MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.
      ) classifies REP34 and related proteins as a non-peptidase homologue of the M14 family. Given the observation that putatively secreted F. tularensis proteins induce the REP phenomenon, which is required for long term survival of the pathogen within amoeba, and the implications of other M14 enzymes in virulence, we hypothesized that REP34 does indeed possess an important, yet unknown function. We surmised that a three-dimensional model of REP34 at atomic resolution would aid in devising experiments to test the function and ultimately may provide insights into the role of REP34 during interactions with phagocytic cells.

      EXPERIMENTAL PROCEDURES

       Identification of REP Proteins

      F. tularensis proteins were identified from bacteria-amoeba coculture subfractions, as described previously (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ). Briefly, subfractions found to induce the highest levels of REP were trypsin-digested using standard methods and were analyzed commercially by ProtTech, Inc. (Norristown, PA) using liquid chromatography tandem mass spectrometry (LC-MS-MS). Single polypeptide coverage for each protein was reported for each protein, resulting in 98% confidence. Proteins were identified from LC-MS-MS data using RefSeq from GenBankTM.

       Francisella Growth and Deletion Mutagenesis

      Francisella strains were grown overnight in Mueller-Hinton broth (Difco) and plated onto tryptic soy agar as described previously (
      • Brotcke A.
      • Weiss D.S.
      • Kim C.C.
      • Chain P.
      • Malfatti S.
      • Garcia E.
      • Monack D.M.
      Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis.
      ). The NOV Δ0149 knock-out mutant was produced as described previously (
      • Brotcke A.
      • Weiss D.S.
      • Kim C.C.
      • Chain P.
      • Malfatti S.
      • Garcia E.
      • Monack D.M.
      Identification of MglA-regulated genes reveals novel virulence factors in Francisella tularensis.
      ). Briefly, a Topo-TA vector (Invitrogen) was used as a surrogate to produce the gene fragment, which was subsequently cloned into a pACYC184 vector in line with a pFNLTP7 kanamycin resistance cassette (
      • Maier T.M.
      • Casey M.S.
      • Becker R.H.
      • Dorsey C.W.
      • Glass E.M.
      • Maltsev N.
      • Zahrt T.C.
      • Frank D.W.
      Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages.
      ). The Δ0149 mutant was selected on modified Mueller-Hinton agar supplemented with kanamycin (15 µg/ml), and gene deletion was confirmed by sequence analysis.

       A. castellanii Infection Assays

      Amoeba were co-infected with F. tularensis, as described previously (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ). Briefly, amoeba cultures grown axenically in PYG broth were seeded in 6-well culture plates (Costar) and incubated overnight at 23 °C in the dark. The PYG was replaced with high salt buffer, and the amoeba was acclimated to 37 °C for 30 min prior to infection. A. castellanii were then infected with NOV overnight cultures at a multiplicity of infection of 10 in triplicate and incubated at 37 °C and 5% CO2 for 30–60 min. The number of cysts and trophozoites were counted in three random fields of view per well using a light microscope; typically, ∼100–200 amoebae were counted in each view.

       Macrophage Infection Assays

      THP-1 cells (ATCC TIB-202) were grown in DMEM (Invitrogen) supplemented with 10% FBS at 37 °C in 24-well tissue culture plates (Costar), similarly to amoeba infection experiments described elsewhere (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ). Briefly, THP-1 cells were seeded at a concentration of 2 × 105 cells/well and infected with NOV overnight cultures at a multiplicity of infection of 10. After co-incubation for 30 min, the cells were washed and incubated with fresh DMEM plus 100 µg/ml gentamicin for either 2 h (“entry”) or 24 h (“proliferation”) at 37 °C and 5% CO2. The cells were then washed once to remove gentamicin and lysed by incubation with 1% saponin (Sigma-Aldrich) for 5 min. Dilutions were plated on supplemented Mueller-Hinton agar to determine viable intracellular cfu counts for the initial inoculum (cfuin), after 2 h (cfu2h), and after 24 h (cfu24h). Entry is expressed as the fraction of cfu2h compared with cfuin.
      Entry=cfu2hcfuin
      (Eq. 1)


      Proliferation is expressed as the fraction of cfu2h compared with cfu2h.
      Proliferation=cfu24hcfu2h
      (Eq. 2)


       REP34 Cloning and Expression

      The NOV gene, FTN_0149, encoding REP34 was cloned into a modified pETBlue expression vector, pETBlueER (Novagen/Merck KGaA, Darmstadt, Germany) by directed cloning (NdeI and BamHI restriction sites). pETBlueER was constructed by replacing the NcoI-XhoI fragment from pETBlue-2 with a 22-bp fragment (5′-CATATGAGAGAGAGAGGGATCC-3′), creating an NdeI-BamHI MCS. The XhoI site is maintained immediately 3′ of the new 22-bp fragment, but the NcoI site is disrupted by blunting with mung bean polymerase (New England Biolabs, Ipswich, MA).
      Both native and selenomethionine REP34 were overexpressed in BL21(DE3) Escherichia coli. Briefly, an overnight starter culture from a single colony was overexpressed in LB, 0.1 mg/liter ampicillin, 37 °C, 220 rpm in shaker flasks and subjected to 1.0 mm isopropyl β-d-1-thiogalactopyranoside induction at A600 = 0.6 for 2 h, pelleted at 4 °C, and stored at −20 °C. Selenomethionine REP34 was produced using the Overnight Express Autoinduction System 2 (Novagen) supplemented with 25 mg/ml l-selenomethionine. Cell pellets were resuspended in buffer A (0.05 m Na+/K+ phosphate, pH 8.0, 0.3 m NaCl, 0.01 m imidazole) and homogenized on ice using an Emulsiflex-C5 (Avestin Inc., Ottawa, Canada). Lysates clarified by centrifugation at 8000 × g for 20 min were purified by nickel-nitrilotriacetic acid affinity chromatography on a 5-ml HisTrap FF column (GE Healthcare) by first washing with buffer A plus 0.02 mm imidazole, followed by elution in buffer A supplemented with 0.25 m imidazole. Fractions in the elution peak were further purified by Superdex-200 size exclusion chromatography (GE Healthcare) in 0.01 m HEPES, pH 7.5, 0.05 m NaCl. The dominant peak, judged >90% pure by SDS-PAGE, was concentrated to 9 mg/ml, flash-frozen in liquid N2, and stored at −80 °C. REP34 + Zn2+ samples were supplemented with 1 mm ZnCl2 prior to performing crystal trials. REP34 site-directed mutants were produced using a modified site-directed mutagenesis protocol (
      • Zheng L.
      • Baumann U.
      • Reymond J.
      An efficient one-step site-directed and site-saturation mutagenesis protocol.
      ) (Stratagene, La Jolla, CA) and cloned into pET24b+ (Novagen). Expression and purification proceeded as described for WT REP34, except that kanamycin at 0.05 mg/liter was substituted for ampicillin.

       REP34 Crystallization and X-ray Structure Determination

      Native and selenomethionine REP34 were screened for crystallization against ∼300 random conditions designed by CRYSTOOL (
      • Segelke B.W.
      Efficiency analysis of sampling protocols used in protein crystallization screening.
      ) in 96-well sitting drop vapor diffusion experiments pipetted with the HYDRA+1 (Robbins Scientific, Sunnyvale, CA). Optimization was first achieved using more stringent CRYSTOOL parameters in 96 wells. Further optimization proceeded in manually prepared hanging-drop vapor diffusion trials (
      • McPherson Jr., A.
      The growth and preliminary investigation of protein and nucleic acid crystals for X-ray diffraction analysis.
      ) consisting of 0.5 ml of well solution containing 20% (w/v) polyethylene glycol 4000, 0.1 m sodium acetate, pH 4.7–5.3, and drops of 1 µl each of protein and well solution at room temperature. Long, icicle-like, heavily twinned crystals appeared overnight with dimensions as large as ∼300 × 50 × 30 µm. Crystals were embedded in Paratone-N (Hampton Research, Hayward, CA) and plunged into liquid N2. X-ray data were collected at the Stanford Synchrotron Radiation Laboratory beamline 12-2 (Pilatus 5M detector) and Advanced Photon Source beamlines GM/CA 23D-D (MARmosaic 300 detector) and Northeastern Collaborative Access Team beamline 24-ID (Pilatus 5M detector) using the microbeam (20 × 20 × 20 µm) (
      • Fischetti R.F.
      • Xu S.
      • Yoder D.W.
      • Becker M.
      • Nagarajan V.
      • Sanishvili R.
      • Hilgart M.C.
      • Stepanov S.
      • Makarov O.
      • Smith J.L.
      Mini-beam collimator enables microcrystallography experiments on standard beamlines.
      ) at 100 K. Microfocus was essential to collect single crystal diffraction data, which was 90% complete to 2.7 Å resolution. Data were processed using XDS (
      • Kabsch W.
      Xds.
      ), and initial phases were obtained using a single wavelength anomalous diffraction (SAD) data set collected at the selenium edge (12.6 keV, 0.979 Å). Four of the five selenium atoms (with the exception of the N-terminal Met) were identified per molecule using SHELXC/D/E (
      • Sheldrick G.M.
      Experimental phasing with SHELXC/D/E: combining chain tracing with density modification.
      ), and an initial model was built using BUCCANEER (
      • Cowtan K.
      The Buccaneer software for automated model building.
      ). Four REP34 molecules were identified in the asymmetric unit.
      Zn2+ co-crystal SAD data (9.66 keV, 1.284 Å) collected from a native REP34 crystal extended the resolution to 2.05 Å (2.4 Å with Friedel pairs unmerged). These data, 95% complete, were processed in HKL2000/3000 (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ). One zinc ion was identified per molecule using SAD/molecular replacement phasing in PHASER with the initial model as the search criteria (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ). Model refinement was carried out in PHENIX by fitting atomic coordinates and atomic displacement parameters individually with 4-fold non-crystallographic symmetry torsion restraints (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.-W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). Manual model building was performed with the Coot graphical user interface (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) using simulated annealing composite omit maps calculated in PHENIX and Shake&wARP maps (
      • Reddy V.
      • Swanson S.M.
      • Segelke B.
      • Kantardjieff K.A.
      • Sacchettini J.C.
      • Rupp B.
      Effective electron-density map improvement and structure validation on a Linux multi-CPU web cluster: the TB Structural Genomics Consortium Bias Removal Web Service.
      ) to reduce model bias. The final model consists of four REP34 chains of total root mean square deviation <0.1 Å that, when combined, contain REP34 residues 4–303 (excluding 242–254), four Zn2+ ions, four acetate ions, and 246 water molecules. The final coordinates and structure factors for REP34 + Zn2+ were deposited in the PDB under accession code 4OKO.

       Structural Analysis

      Structural homologues for REP34 were identified using the 3D-BLAST search algorithm (
      • Tung C.H.
      • Huang J.W.
      • Yang J.M.
      κ-α plot derived structural alphabet and BLOSUM-like substitution matrix for rapid search of protein structure database.
      ). Structural alignments were performed with the MatchMaker function in CHIMERA (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera: a visualization system for exploratory research and analysis.
      ) using the default parameters. Solvent-accessible pockets were calculated using the CASTp online server (
      • Dundas J.
      • Ouyang Z.
      • Tseng J.
      • Binkowski A.
      • Turpaz Y.
      • Liang J.
      CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.
      ) by submitting single chain coordinates for REP34, CPT (PDB code 1OBR (
      • Teplyakov A.
      • Polyakov K.
      • Obmolova G.
      • Strokopytov B.
      • Kuranova I.
      • Osterman A.
      • Grishin N.
      • Smulevitch S.
      • Zagnitko O.
      • Glalperina O.
      • Matz M.
      • Stepanov V.
      Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.
      )), and CPB (PDB code 1Z5R (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      )) with solvent molecules removed. Structures were prepared for Poisson-Boltzman electrostatic potential calculations using PDB entry 2PQR (
      • Dolinsky T.J.
      • Nielsen J.E.
      • McCammon J.A.
      • Baker N.A.
      PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations.
      ), and the calculations were performed using APBS (
      • Baker N.A.
      • Sept D.
      • Joseph S.
      • Holst M.J.
      • McCammon J.A.
      Electrostatics of nanosystems: application to microtubules and the ribosome.
      ).

       Carboxypeptidase Activity Spectrophotometry

      Hydrolysis of hippuryl-l-arginine (Sigma-Aldrich) was monitored by change in absorbance at 254 nm, as described previously (
      • Folk J.E.
      • Piez K.A.
      • Carroll W.R.
      • Cladner J.A.
      Carboxypeptidase B: purification of characterization of the porcine enzyme.
      ), with slight modification for 96-well plates, using a BioTek Synergy HT microplate reader (Winooski, VT). Briefly, 3 µl of protein (recombinant REP34 (rREP34) WT and point mutants) was added to each well (final protein concentration in the well was 1.5 µg/ml), and 97 µl of hippuryl-l-Arg prepared fresh in reaction buffer (0.025 m Tris-HCl, pH 7.65, 0.1 m NaCl, 1.0 mm ZnCl2) was added and mixed with a multichannel pipette, mixing twice before measurement. For REP34 concentration dependence experiments, 10 µl of protein was added to each well, followed by 190 µl of hippuryl-l-Arg and mixing. Absorbance was measured every 7–10 s for 5 min, and Vo for each run was calculated. Michaelis-Menten kinetic parameters were determined using non-linear regression analysis in Microsoft Excel.

       Inhibition of REP34

      The carboxypeptidase B inhibitor 2-guanidinoethylmercaptosuccinic acid (GEMSA) was purchased from Calbiochem (EMD Millipore, Billerica, MA). 1 mg/ml WT REP34 was incubated with a 10-fold molar excess of GEMSA in buffer B plus 1.0 mm ZnCl2 at room temperature for 1 h. The molecular masses of REP34 both with and without GEMSA were analyzed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry on a Voyager-DE system (PerSpective Biosystems).

      RESULTS

       REP34 Contributes to Amoeba Encystment

      NOV genes corresponding to proteins believed to be responsible for REP (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ) were identified using LC-MS-MS and are presented in Table 1. We constructed a knock-out strain of NOV lacking the FTN_0149 gene (Δ0149) and infected A. castellanii with both Δ0149 and wild-type NOV (Fig. 1A). The Δ0149-infected amoebae show significantly less (p < 0.0001) encystment compared with wild type NOV-infected amoeba. Although 86 ± 11% of amoebae encysted in response to NOV, only 9 ± 4% formed cysts in response to Δ0149. Significant (p < 0.005) encystment was restored by the addition of 10 µg/ml recombinant REP34 (rREP34) to the knock-out strain because 21 ± 8% of amoeba encysted under these conditions. A comparable fraction of amoeba formed cysts for both the addition of rREP34 alone and a phosphate-buffered saline (PBS) control, 4.3 ± 0.3 versus 5 ± 1%, respectively. Thus, our data suggest that FTN_0149 is important for amoebic encystment during F. tularensis infection, but the exogenous FTN_1049 gene product, rREP34, is only modestly able to complement the encystment-inducing activity and, in the absence of other F. tularensis components, is unable to induce REP.
      TABLE 1Identification of F. tularensis proteins that mediate REP in A. castellanii
      Locus IDProtein designation
      Based on size (kDa).
      Putative functionNo. of peptides
      Number of peptides sequenced by nano-LC-MS-MS.
      FTN_0149REP34Hypothetical protein1
      FTN_0506REP13Glycine cleavage system H protein1
      FTN_0550REP37Predicted inner membrane peptidase1
      FTN_0841REP24ThiJ/Pfpl family protein1
      FTN_0962REP30Hypothetical protein1
      FTN_1296REP11Ribosomal subunit interface protein1
      FTN_1331REP42Phosphoglycerate kinase1
      a Based on size (kDa).
      b Number of peptides sequenced by nano-LC-MS-MS.
      Figure thumbnail gr1
      FIGURE 1REP34 is implicated in F. tularensis host cell invasion. A, ability of NOV strains to induce amoeba to encyst, where Fraction Encysted = cysts/total. Error bars, S.D. of three independent technical replicates (three field-of-view counts, n = 3–9). B, ability of F. tularensis strains to enter (Fraction Entry; left scale and black bars) and subsequently survive (Fraction Proliferate; right scale and gray bars) in human monocyte hosts. Histograms and error bars, mean and S.D. of cfu in three independent counts (n = 3). Significance was determined by Student's t test (***, p < 0.0001; **, p < 0.005; *, p < 0.05). C, crystal structure of REP34 rendered as ribbons in the standard view for funnelins (
      • Gomis-Rüth F.X.
      Structure and mechanism of metallocarboxypeptidases.
      ) and colored by secondary structure: helices (orange), strands (green), and loops (purple). The Zn2+ is represented as a cyan sphere. N and C termini are indicated. D, REP34 depicted as a translucent molecular surface, colored as in B and rotated ∼90° around the x axis. All figures were prepared in CHIMERA (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera: a visualization system for exploratory research and analysis.
      ).

       REP34 Contributes to F. tularensis Entry into Human Monocytes

      Given the hypothesis that protozoa may serve as “training grounds” for intracellular pathogens and the observation that Δ0149 is significantly impaired in its ability to induce REP in A. castellanii (Fig. 1A), we hypothesized that the Δ0149 strain might also exhibit a reduced ability to infect human macrophages. To test this hypothesis, we infected human monocytes with NOV, Δ0149, and LVS. Two time points were taken to investigate the ability of F. tularensis to enter (2 h) and to replicate (24 h) in human monocytes. Host cell entry was significantly reduced (p < 0.0001) in Δ0149 because this mutant strain entered monocytes at a rate of 9.7 ± 0.9% versus 53 ± 14% for NOV. The Δ0149 knockout had less of an effect (p < 0.05) on F. tularensis proliferation in monocytes because Δ0149 increased at a rate of 620 ± 60% over 22 h post-entry versus 1100 ± 300% for NOV. For comparison, LVS, which lacks two REP-encoding genes and does not induce amoeba encystment (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ), can enter macrophages similarly to NOV, 48 ± 18%, but proliferation within host cells was severely reduced, 41 ± 32%. Thus, the data suggest that FTN_0149 is involved in the entry of human monocytes but has a marginal effect on either bacterial replication or survival.

       REP34-Zn2+ Co-crystallization

      Purified rREP34 formed snowflake-like crystals with multiple lattices, which proved recalcitrant to single crystal diffraction studies. Single-crystal data could only be collected with a microbeam x-ray source on larger crystal clusters grown from larger volume drops. An initial REP34 structure was determined by Se-SAD phasing of selenomethionine-derived REP34 to 2.7 Å resolution with a triclinic lattice. These crystals were grown in the presence of 5–10 mm EDTA, and evidence for metal cofactors was absent from both x-ray fluorescence measurements and electron density. Because the resulting partially built structure resembled the conical metallocarboxypeptidase funnelin fold and included the expected HXXE Zn2+ coordination motif for these enzymes, we subsequently crystallized REP34 with 1 mm ZnCl2 and without EDTA. The zinc-containing REP34 crystals diffracted x-rays to 2.05 Å (Table 2), had nearly identical unit cell dimensions as the crystals grown in the presence of EDTA, and gave x-ray fluorescence signatures consistent with the presence of zinc. A new data set collected at the zinc edge (9.66 keV) was used to determine the zinc-containing REP34 structure by molecular replacement using the zinc-free structure as the search model. Continuous electron density covering almost all four copies of REP34 in the asymmetric unit was evident, and residues 4–303 were placed, except for a disordered loop, 233–242, which is disordered in all four copies in the asymmetric unit (Fig. 1, C and D).
      TABLE 2Data collection and refinement statistics
      REP34 (−Zn2+)
      Data collected from a single selenomethionine crystal.
      REP34 (+Zn2+)
      Data collected from a single native crystal.
      PDB code4OKO
      Data collection
      Wavelength (keV)12.69.66
      Space groupP1P1
      Cell dimensions
      a, b, c (Å)62.79, 63.53, 82.5862.71, 63.31, 81.69
      α, β, γ (degrees)70.37, 84.45, 83.0370.36, 84.34, 82.53
      Resolution (Å)77.64–2.73 (2.80–2.73)
      Values for the highest resolution shell are shown in parenthesis.
      22.00–2.05 (2.09–2.05)
      Values for the highest resolution shell are shown in parenthesis.
      Rp.i.m.0.085 (0.307)0.078 (0.437)
      II7.3 (2.3)9.0 (2.1)
      Completeness (%)89.9 (82.2)94.9 (83.1)
      Redundancy3.1 (3.1)2.6 (2.6)
      Phasing
      Figure of merit (FOM)0.468
      Figure of merit after Buccaneer0.808
      Model map CC0.727
      Refinement
      Resolution (Å)77.63–2.73 (2.80–2.73)21.54–2.05 (2.10–2.05)
      No. of reflections28,542 (1889)68,913 (4503)
      Rwork/Rfree21.3/23.3 (26.1/32.1)
      No. of atoms
      Protein77369180
      Ligand020
      Water0240
      B-Factors
      Protein42.4
      Ligand47.4
      Water40.2
      Root mean square deviations
      Bond lengths (Å)0.004
      Bond angles (degrees)0.849
      Molprobity statistics
      All-atom clashscore2.05
      Ramachandran favored97.9
      Ramachandran allowed2.1
      Ramachandran outliers0.0
      Overall score1.10
      a Data collected from a single selenomethionine crystal.
      b Data collected from a single native crystal.
      c Values for the highest resolution shell are shown in parenthesis.

       REP34 Possesses an α/β Hydrolase-like Fold

      REP34 is structurally homologous to Zn2+-carboxypeptidases with funnelin-like architecture, possessing the α88 topology of α/β hydrolases. To simplify analysis and discussion, here we use CPT from Thermoactinomyces vulgaris (PDB code 1OBR (
      • Teplyakov A.
      • Polyakov K.
      • Obmolova G.
      • Strokopytov B.
      • Kuranova I.
      • Osterman A.
      • Grishin N.
      • Smulevitch S.
      • Zagnitko O.
      • Glalperina O.
      • Matz M.
      • Stepanov V.
      Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.
      )) as a representative homologous bacterial carboxypeptidase T and porcine pancreatic CPB (PDB code 1Z5R (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      )) as a representative carboxypeptidase B. Despite sequence identities between REP34 and CPT or CPB of less than 15%, both CPT and CPB align well structurally with REP34 (backbone root mean square deviations of ∼2.5 Å) (Fig. 2A). Canonical Zn2+-carboxypeptidases possess three principal structural elements that contribute to substrate recognition and binding. First, a long loop connecting α3 and α4 (Lα3α4) forms the outermost lip of the active site, providing the solvent-exposed rim of the funnel. Second, the active site residues surrounding the Zn2+ coordination site catalyze the hydrolysis chemistry of the enzyme. Third, the P1′ substrate recognition site is lined with residues that provide shape and chemical complementarity to the C-terminal side chain of a substrate, giving rise to the substrate specificity of the enzyme.
      Figure thumbnail gr2
      FIGURE 2Comparison of REP34 with two carboxypeptidase structural homologues. Sequences and structures were aligned using MatchMaker in CHIMERA (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera: a visualization system for exploratory research and analysis.
      ); secondary structure (colored as in ) and residue numbering convention correspond to REP34 primary sequence numbering. A, stereo structural alignments for REP34 (purple), CPT from T. vulgaris (PDB code 1OBR (
      • Teplyakov A.
      • Polyakov K.
      • Obmolova G.
      • Strokopytov B.
      • Kuranova I.
      • Osterman A.
      • Grishin N.
      • Smulevitch S.
      • Zagnitko O.
      • Glalperina O.
      • Matz M.
      • Stepanov V.
      Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.
      ), blue), and porcine pancreatic CPB (PDB code 1Z5R (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      ), cyan). REP34 α0 and α4′ as well as CPB/T Lα3α4 are indicated; REP34 Zn2+ is rendered as a sphere (gold). B, topology map for the enzymes depicted in A. Conserved helices and strands are colored as in , whereas those unique to REP34 are colored purple. N and C termini are indicated. C, sequence alignment and secondary structure assignments for REP34, CPT, and CPB. Active site and Zn2+-coordinating residues are highlighted (red and blue, respectively), and REP34 α0 and α4′ are identified with stars (yellow).
      The REP34 structure exhibits both similar and unique features relative to its closest structural homologues. When observed from the standard view for funnelins (
      • Gomis-Rüth F.X.
      Structure and mechanism of metallocarboxypeptidases.
      ), the active site Zn2+ is positioned at the C terminus of the central four-strand parallel sheet, as expected (Fig. 1C). In contrast, REP34 also contains additional structural elements, namely an N-terminal helix, α0,
      Secondary structure elements are numbered according to funnelin convention.
      and an insertion helix, α4′ (Fig. 2, A and B) on the side opposite the central parallel sheet. While the canonical funnelin loop Lα3α4 that defines the right edge of the substrate-binding cleft is quite long, containing ∼60 residues, the homologous loop in REP34 is much shorter, containing only ∼20 residues (Fig. 2C). Furthermore, REP34 α6 is positioned away from the active site cleft, whereas the corresponding helix and preceding loop in metallocarboxypeptidases forms a wall of the active site and donates a conserved tyrosine, which stabilizes the P1′ amino acid of the substrate. Taken together, this combination of the two additional helices, substantially shorter Lα3α4, and rearranged α6 produces a distinct cleft leading to the Zn2+, when compared with the “funnel” shape of recognized metallocarboxypeptidases (Fig. 1D). Thus, REP34 possesses the central α/β hydrolase fold indicative of metallocarboxypeptidases but has unique structural features that probably redefine how the enzyme interacts with its target peptide.
      As a result of these structural differences surrounding the active site cleft, REP34 possesses a larger substrate cavity compared with CPT and CPB (Fig. 3). This cavity occupies a volume of 1793 Å3 for REP34, which is more than twice as large as the 732-Å3 cavity in CPB and more than 3 times the 432-Å3 volume for CPT, as calculated by CASTp (
      • Dundas J.
      • Ouyang Z.
      • Tseng J.
      • Binkowski A.
      • Turpaz Y.
      • Liang J.
      CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.
      ). Factors that appear to contribute to the larger cavity include a wider rim at the protein surface, a wider internal cavity where the putative substrate is expected to reside during hydrolysis, and an additional narrow cavity extending through the entire molecule. The cavity linking the substrate-binding pocket to the rest of the pocket on the opposite side of REP34 is too narrow to fit a polypeptide. Overall, REP34 contains a large substrate-binding cavity that appears accessible for polypeptide binding and subsequent hydrolysis.
      Figure thumbnail gr3
      FIGURE 3Substrate binding pocket comparison for REP34 and related carboxypeptidases. Shown are surface representations of REP34 (purple, left), CPT (PDB 1OBR (
      • Teplyakov A.
      • Polyakov K.
      • Obmolova G.
      • Strokopytov B.
      • Kuranova I.
      • Osterman A.
      • Grishin N.
      • Smulevitch S.
      • Zagnitko O.
      • Glalperina O.
      • Matz M.
      • Stepanov V.
      Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.
      ), blue, middle), and CPB (PDB 1ZG7 (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      ), cyan, right), where substrate binding pocket surfaces (gold) are computed using the CASTp server (
      • Dundas J.
      • Ouyang Z.
      • Tseng J.
      • Binkowski A.
      • Turpaz Y.
      • Liang J.
      CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues.
      ). The top views are of each molecule looking into the pocket, and the bottom views are 90° rotations around the x axis with a clipping plane placed at the center of the pocket. For CPT and CPB, the top view is the standard view, and for REP34, the view is ∼90° rotation around the x axis relative to the standard view. CPB contains a bound inhibitor (sticks, pink) to aid in visualization of the binding pocket-substrate interaction.

       REP34 Active Site

      A Zn2+ coordination site represents the centerpiece of the REP34 active site, consisting of side chains His86, Glu89, and His171 and a coordinated water molecule in a distorted tetrahedral geometry (Fig. 4A). Anomalous difference maps from SAD data collected at the zinc edge pinpoint the metal ion's position. The map clearly reveals the location of the Zn2+, and prominent 2FoFc density corresponding to the coordinating side chains is also evident. Therefore, REP34 contains a Zn2+ coordinated by an HXXEH motif typical of metallocarboxypeptidases.
      Figure thumbnail gr4
      FIGURE 4Active site of REP34. A, close-up view of the Zn2+ binding site colored by atom (carbon (purple), nitrogen (blue), oxygen (red), and Zn2+ (cyan)), where Zn2+ and the coordinated water molecule are depicted as spheres. Electron density for a simulated annealing composite omit map calculated at the end of refinement to 2.05 Å resolution and contoured to 1.0σ is depicted as a mesh (gray). An anomalous difference map revealing the location of the Zn2+ ion and calculated at the end of refinement to 2.4 Å resolution is represented by a mesh (orange) contoured to 8.5σ. B, MatchMaker-generated structural alignment of the active sites for REP34 (purple) and CPB (PDB Code 1Z5R (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      ), yellow), with atoms colored as in A. Residue numbering corresponds to REP34, except for CPB Tyr-248. C, MatchMaker-generated overlay of REP34 with CPA4-cleaved hexameric peptide (PDB code 2PCU (
      • Bayés A.
      • Fernández D.
      • Solà M.
      • Marrero A.
      • García-Piqué S.
      • Avilés F.X.
      • Vendrell J.
      • Gomis-Rüth F.X.
      Caught after the act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.
      ), green) and CPT-bound sulfamoyl arginine (PDB code 4GM5, orange). The P1′ residue of the former and the sulfamoyl group of the latter bound substrates are omitted for clarity. The composite omit electron density as in A, contoured to 1.6σ for the ordered acetate bound to REP34, is depicted as a mesh (gray).
      In addition to the residues coordinating the Zn2+, funnelins have five other conserved residues involved in catalysis (Fig. 2C). Using the standard CPA/B sequence numbering, these include Glu270, which polarizes the nucleophilic water and donates a hydrogen to the amino group of the P1′ leaving group; Asn144 and Arg145, which stabilize the P1′ C terminus during catalysis; Tyr-248, which stabilizes the P1 amide and the P1′ C terminus; and Arg-127, which polarizes the carbonyl of the P1 amino acid during the tetrahedral intermediate. REP34 residues Glu-272, Asn-138, Arg-139, and Arg-129 overlap quite well with CPA/B residues Glu-270, Asn-144, Arg-145, and Arg-127, respectively (Fig. 4B). The loop containing CPA/B Tyr-248 is largely absent from REP34 (Fig. 2C); however, Tyr-88 may be a compensatory substitution in REP34, affording at least some of the intermediate stabilization that Tyr-248 provides in CPA/B. In sequences homologous to REP34 (∼50% identical), this Tyr, as well as flanking residues that include His and Glu Zn2+ coordinators, is absolutely conserved. REP34 Tyr-88 is positioned on the opposite side of the active site cleft compared with Tyr-248 in other funnelins. Despite the rearrangement, Tyr-88 remains well positioned to contact the P1 amide as well as to polarize the P1 carbonyl. This is evident by aligning REP34 with two structures of funnelins bound to substrates, human carboxypeptidase A4 (CPA4) in complex with a hexapeptide post-bond cleavage (PDB code 2PCU (
      • Bayés A.
      • Fernández D.
      • Solà M.
      • Marrero A.
      • García-Piqué S.
      • Avilés F.X.
      • Vendrell J.
      • Gomis-Rüth F.X.
      Caught after the act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.
      )) and a CPT-sulfamoyl arginine co-complex. However, the presence of α4′ in REP34 severely clashes with the polypeptide, implying that a substrate must adopt a different conformation when bound to REP34, as compared with other structurally related funnelin substrate complexes (Fig. 4C). To test this hypothesis, we incubated REP34 with GEMSA, a small molecule known to inhibit CPB enzymes. Subsequent mass spectrometry analysis revealed that both the GEMSA-incubated and free enzyme samples were the same mass, indicating that GEMSA does not covalently modify REP34. Albeit a negative result, this is consistent with the structural differences that impart a unique substrate-binding pocket in REP34 relative to CPB, occluding the binding of the inhibitor GEMSA to REP34.
      REP34 was crystallized in the presence of 0.1 m sodium acetate, and strong electron density consistent with a single acetate anion is present in the active site of each REP34 molecule in the asymmetric unit (Fig. 4C). Acetate resembles the C terminus of peptides, and the high concentration in the crystallization condition probably resulted in it being trapped in the active site of REP34. The oxygen atoms of the acetate carboxyl interact with the side chains of REP34 Arg-129, Asn-138, and Arg-139. These residues correspond to CPA/B amino acids that participate in P1′ stabilization in a similar manner. Furthermore, Cα alignment of CPA/B/T structures containing inhibitors or molecules with either cleaved P1′ amino acids (
      • Bayés A.
      • Fernández D.
      • Solà M.
      • Marrero A.
      • García-Piqué S.
      • Avilés F.X.
      • Vendrell J.
      • Gomis-Rüth F.X.
      Caught after the act: a human A-type metallocarboxypeptidase in a product complex with a cleaved hexapeptide.
      ) or P1′ mimics (
      • Adler M.
      • Bryant J.
      • Buckman B.
      • Islam I.
      • Larsen B.
      • Finster S.
      • Kent L.
      • May K.
      • Mohan R.
      • Yuan S.
      • Whitlow M.
      Crystal structures of potent thiol-based inhibitors bound to carboxypeptidase B.
      ,
      • Cho J.H.
      • Kim D.H.
      • Chung S.J.
      • Ha N.C.
      • Oh B.H.
      • Yong Choi K.
      Insight into the stereochemistry in the inhibition of carboxypeptidase A with N-(hydroxyaminocarbonyl)phenylalanine: binding modes of an enantiomeric pair of the inhibitor to carboxypeptidase A.
      ,
      • Reverter D.
      • Fernández-Catalán C.
      • Baumgartner R.
      • Pfänder R.
      • Huber R.
      • Bode W.
      • Vendrell J.
      • Holak T.A.
      • Avilés F.X.
      Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2.
      ) (PDB entry 4GM5) places these carboxyl groups in close proximity to the bound acetate ion in the REP34 structure. Therefore, this trapped acetate is consistent with the P1′ C terminus of a cleaved target peptide in the product-bound state.

       Carboxypeptidase Activity of REP34

      From the crystal structure, it appears that REP34 could be an active carboxypeptidase, despite the MEROPS annotation, so we assayed for carboxypeptidase activity using rREP34. We measured the change in absorbance at 254 nm caused by the release of hippuric acid from the model substrates hippuryl-l-Phe and hippuryl-l-Arg for CPA and CPB, respectively. Incubation of REP34 with hippuryl-l-Phe did not result in any appreciable release of hippuric acid over a range of protein concentrations (data not shown). Therefore, we deduced that the protein has little, if any, CPA activity. In the presence of hippuryl-l-Arg, we measured considerable hydrolysis, with absorbance changes similar to previous studies of purified porcine pancreatic CPB (
      • Folk J.E.
      • Piez K.A.
      • Carroll W.R.
      • Cladner J.A.
      Carboxypeptidase B: purification of characterization of the porcine enzyme.
      ). By varying the concentration of REP34 in the reaction, we find that hippuryl-l-Arg hydrolysis is enzyme concentration-dependent, plateauing around 6 µg ml−1 protein, for 1.0 mm substrate (Fig. 5A). Varying the substrate concentration while holding [REP34] at 1.5 µg/ml, we observed a kinetic profile that fits a Michaelis-Menten model for the range of substrate concentrations measured (Fig. 5B). We were unable to measure a constant background signal for hippuryl-l-Arg concentrations higher than 5.0 mm; therefore, these measurements were excluded from further analysis. Furthermore, the calculated Km for these kinetic measurements was ∼18 mm, which is considerably higher than an achievable substrate concentration under these conditions. Although we cannot confirm whether REP34 follows a classical kinetic profile for the model substrate hippuryl-l-Arg, we conclude that REP34 appears to have robust CPB-like activity.
      Figure thumbnail gr5
      FIGURE 5REP34 carboxypeptidase activity. The rate of hydrolysis of the carboxypeptidase B model substrate hippuryl-l-Arg was measured by absorbance change at 254 nm for REP34 concentration dependence (A) and hippuryl-l-Arg concentration dependence (B). The line represents nonlinear regression best fit according to the Michaelis-Menten equation. 1 unit = hydrolysis of 1.0 µmol of hippuryl-l-Arg/min at pH 7.65, 25 °C. C, percentage activity of REP34 active site mutants to hydrolyze hippuryl-l-Arg, as compared with WT REP34. Error bars, S.D. for multiple trials (n = 3–5).
      As a structural homologue of CPB (FIGURE 2, FIGURE 4), we hypothesized that REP34 has a similar catalytic mechanism. To test this hypothesis, we produced alanine point mutations for each of the homologous putative catalytic residues in REP34: Y88A, R129A, N138A, R139A, and E272A. We then measured the ability of these mutants to hydrolyze hippuryl-l-Arg relative to WT (Fig. 5C). For these experiments, the concentration of initial hippuryl-l-Arg was kept constant at 1.0 mm, and hydrolysis was determined as the maximum change in absorbance. The REP34 mutants E272A and Y88A showed the greatest loss in activity, resulting in 18 ± 2 and 23 ± 13% activities relative to WT, respectively. The REP34 substitutions R139A and N138A have activities of 33 ± 7 and 49 ± 16%, respectively. In contrast, the mutant R129A hydrolyzed hippuryl-l-Arg similarly to WT, with a 97 ± 15% activity. Therefore, residues Glu-272 and Tyr-88 contribute significantly to REP34 catalysis, whereas Arg-129 appears to perform a nonessential role in catalysis.

       Identification of Putative P1′ Recognition Site

      The metallocarboxypeptidases confer substrate specificity by shape and chemical complementarity to the P1′ residue in the substrate recognition S1′ site. The structural rearrangement of REP34 results in a binding pocket that probably accommodates substrate differently than other funnelins (FIGURE 3, FIGURE 4C). Given that we demonstrate robust CPB-like activity for REP34 (Fig. 5), we would expect REP34 to possess a corresponding S1′ site equipped with at least one negative charge. Indeed, close to the active site are the acidic residues Glu-256 and Asp-177 (Fig. 6A), which contribute to two different cleft surfaces predicted to be strongly electronegative (Fig. 6B). The Glu-256 site is lined with additional residues Val-205, Cys-251, Leu-258, and Thr-270, which are similar in chemistry to the corresponding S1′ site residues Ala-250, Thr-268, and Leu-203 in CPB (Fig. 6C). Residue Asp-177 is flanked by Thr-173 as well as the acidic residues Asp-175, Asp-178, and Asp-276. REP34 Tyr-203 provides additional polar surface to both putative S1′ sites (Fig. 6A).
      Figure thumbnail gr6
      FIGURE 6S1′ recognition site in REP34. REP34 and CPB (PDB 1Z5R (
      • Teplyakov A.
      • Polyakov K.
      • Obmolova G.
      • Strokopytov B.
      • Kuranova I.
      • Osterman A.
      • Grishin N.
      • Smulevitch S.
      • Zagnitko O.
      • Glalperina O.
      • Matz M.
      • Stepanov V.
      Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.
      )) are aligned and colored as in . The region of interest is enclosed in the box. A, putative S1′ recognition sites in REP34, with the molecular surface colored gray. B (top), electrostatic surface rendering of REP34, where computed negative, neutral, and positive charges are colored red, white, and blue, respectively. The active site cleft is boxed and magnified (bottom), where the positions of the Zn2+, Glu-256, and Asp-177 are indicated. A clipping plane above the cleft and approximately parallel to the page was applied for clarity. C, comparison of the S1′ sites in REP34 and CPB, with the molecular surface for CPB colored gray. CPB Zn2+ is colored dark gray; all labels correspond to CPB elements except for REP34 α4′.
      Distance geometry homology suggests that Glu-256 is more consistent with the P1′ complementing residue. The distances from the δ- and γ-carbons for REP34 Glu-256 and Asp-177 to the Zn2+ are 12.0 and 6.8, respectively, whereas Asp-175, Asp-178, and Asp-276 are more remote (>13 Å). The corresponding distances for the P1′ basic side chain recognition residues in CPB (Asp-255) and CPT (Asp-260) are 10.5 and 10.1 Å, respectively. Therefore, REP34 Glu-256 appears well positioned to complement a positively charged P1′ residue during substrate binding. Whereas REP34 Asp-177 is considerably closer to the Zn2+ than either aspartic acid in CPB or CPT, it also points more directly toward the Zn2+; thus, it may interact less strongly with a P1′ basic residue.

      DISCUSSION

      Infection assays involving Δ0149 implicate a role for REP34 in both the induction of REP and the entry of F. tularensis in human monocytes (Fig. 1, A and B). As shown in Fig. 1A and elsewhere (
      • El-Etr S.H.
      • Margolis J.J.
      • Monack D.
      • Robison R.A.
      • Cohen M.
      • Moore E.
      • Rasley A.
      Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection.
      ), F. tularensis NOV induces REP in amoeba, and Δ0149 NOV strain has a significantly reduced ability to induce this phenotype. The ability of the FTN_0149 gene to induce encystment may be more complicated than simply expressing and secreting REP34 because rREP34 does not restore REP activity to wild-type NOV levels, and the addition of rREP34 alone did not elicit REP. It is certainly plausible that some of the other REP-inducing genes (Table 1) may also encode for effector proteins, and the Δ0149 mutant may disrupt the REP machinery in ways supplementary to REP34 expression. Thus, we suspect that REP34 may work in concert with other REP proteins to induce REP.
      It has been suggested that interactions between intracellular pathogens, including F. tularensis, and environmental amoebae might have resulted in these bacteria evolving phagocytic cell evasion tactics (
      • Miyake M.
      • Watanabe T.
      • Koike H.
      • Molmeret M.
      • Imai Y.
      • Abu Kwaik Y.
      Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages.
      ), which may in turn enhance their ability to infect macrophages (
      • Salah I.B.
      • Ghigo E.
      • Drancourt M.
      Free-living amoebae, a training field for macrophage resistance of mycobacteria.
      ). Our data suggest that REP34 plays a significant role in host cell entry but less of a role in either the survival or replication of the pathogen within the host cell (Fig. 1B). Interestingly, the LVS strain, which lacks two of the genes encoding REPs, is capable of entering monocytes similarly to NOV but lacks the ability to survive and replicate. Further research into the targets of REP34 and other REP proteins may shed light on their roles in mediating interactions of F. tularensis with phagocytic cells.
      The crystal structure of REP34 presented here provides preliminary evidence for the carboxypeptidase activity of REP34. The presence of a α/β hydrolase core renders REP34 a close structural homologue of the M14 funnelin family of Zn2+-dependent carboxypeptidases with overall root mean square deviations of ∼2.5 Å despite sequence similarities of less than 15%. The MEROPS peptidase database classifies REP34 and similar proteins as inactive members of the M14 family, probably because a structural rearrangement of the active site results in a displacement of the REP34 counterpart for a catalytically important Tyr (Tyr-248 for CPB and Tyr-255 for CPT). We show that REP34 is, indeed, an active carboxypeptidase (Fig. 5). Furthermore, REP34 Tyr-88, which is highly conserved in the subfamily, may represent a compensatory substitution for the canonical catalytic Tyr. This is consistent with the Y88A mutant showing an ∼80% decrease in carboxypeptidate activity relative to wild type (Fig. 5C).
      Analysis of the REP34 structure provides clues as to the nature of the enzyme's activity and substrate binding. Compared with other funnelins, REP34 has a shorter Lα3α4 and contains additional helices α0 and α4′, giving rise to a larger predicted binding cleft (Fig. 3) and a unique structural scaffolding for the active site (Figs. 4C and 6C). These differences probably result in novel enzyme-substrate interactions. Biochemical data provide strong evidence for CPB-like activity for REP34 (Fig. 5), and the structure provides a basis to predict a S1′ recognition site on the enzyme, which presents negative charges to complement the predicted basic P1′ residue of the target substrate (Fig. 6, A and B). In addition, a trapped acetate ion is present in the active site (Fig. 4C) and probably approximates the C terminus of a P1′ amino acid of the target protein in the post-hydrolysis state. Taken together, these data strongly support the CPB-like activity of REP34 in catalyzing the hydrolysis of a C-terminal basic residue from an unknown target protein.
      Based on proposed catalytic mechanisms for other metallocarboxypeptidases, we developed a schematic mechanism (Fig. 7) for the hydrolysis of a C terminal basic residue that is consistent with the REP34 structure (FIGURE 2, FIGURE 3, FIGURE 4 and 6) and biochemical data (Fig. 5) presented here. Contacts between substrate polypeptide atoms and REP34 residues Asn-138, Arg-139, and Glu-272 as well as the catalytic water and Zn2+ during the catalytic cycle are assumed to be roughly identical to funnelin active site residues described elsewhere (
      • Gomis-Rüth F.X.
      Structure and mechanism of metallocarboxypeptidases.
      ). The exceptions are Tyr-88, which adopts a geometry different from that of its presumed functional homologue in CPB, Tyr-248 (Fig. 4B), and Arg-129, which from mutagenesis experiments appears to be superfluous to the catalytic mechanism (Fig. 5C).
      Figure thumbnail gr7
      FIGURE 7Schematic mechanism of peptide hydrolysis by REP34. Residues are numbered according to REP34 convention (purple) except for CPB/CPA Arg-127 and Tyr-248 (cyan). Substrate P1 and P1′ amino acids are indicated (black). Arrows, movement of electrons; dotted lines, likely stabilization contacts of enzyme with substrate. The mechanism was based on previously described mechanisms for funnelins (
      • Gomis-Rüth F.X.
      Structure and mechanism of metallocarboxypeptidases.
      ), but REP34 Tyr-88 serves a novel role in REP34. Based on structural (FIGURE 2, FIGURE 3, FIGURE 4 and ) and biochemical data (), Tyr-88 may participate in P1 amide and carbonyl stabilization rather than P1 amide and P1′ carboxyl stabilization.
      Structural superposition of CPB and REP34 places CPB Tyr-248 in close proximity to the acetate position in the REP34 structure, which is assumed to mimic the P1′ C terminus (Fig. 4C). This Tyr is presumed to stabilize the polypeptide substrate by contacting both the negative charge of the P1′ carboxyl and the P1 amide. Although Tyr-88 can still potentially stabilize the P1 amide, Tyr-88 is not close enough to interact with the acetate. Instead, Tyr-88 is within hydrogen-bonding distance of the catalytic water. In the transition state, the catalytic water adds to the P1 peptide carbon, forming a tetragonal bond; Tyr-88 would putatively contact the P1 carbonyl position. This positions the Tyr-88 hydroxyl to stabilize the “anionic hole” created by the partial negative charge on the carbonyl following water addition. CPB Arg-127 is expected to provide this polarizing stability in the canonical mechanism; however, the homologous residue in REP34, Arg-129, does not appear to play as critical a role because REP34 R129A activity is not reduced in vitro (Fig. 5C). Given that the solution side chain pKa values of Tyr and Arg are greater than 10, it is not unreasonable for Tyr-88 to provide a positively charged dipole stabilization of the negatively charged substrate carbonyl, which is chemically similar to the role of Arg-127 in CPA/B. Therefore, we propose that this alternative role for REP34 Tyr-88 in stabilizing the substrate P1 carbonyl may compensate for the R129A substitution. A co-crystal structure containing REP34 bound to a substrate mimic would be informative in further defining the catalytic mechanism of the enzyme and provide a template for the rational design of countermeasures against REP34 and REP34-like peptidases in other pathogenic organisms.
      A BLAST search of nonredundant genetic homologues to REP34 reveals a number of bacterial genera that also may produce similar enzymes. Homologous sequences (∼50%) are found in Vibrio species, including the causative agent of cholera, V. cholerae, as well as the primarily marine bacteria Shewanella and Alteromas. Generally, these genes are annotated as either hypothetical proteins or some derivative of zinc-containing metalloenzymes, including hydrolases and desuccinalases; however, all of these sequences contain a conserved Tyr corresponding to REP34 Tyr-88, nestled between Zn2+-coordinating His and Glu residues. In the case of V. cholerae, the putative homologue (gene code VCA0936) is classified as a conserved hypothetical protein and is present on chromosome 2. VCA0936 has been implicated in biofilm production by avirulent V. cholerae strains, which may contribute to the tenacity of these organisms in their marine ecosystems (
      • Mueller R.S.
      • McDougald D.
      • Cusumano D.
      • Sodhi N.
      • Kjelleberg S.
      • Azam F.
      • Bartlett D.H.
      Vibrio cholerae strains possess multiple strategies for abiotic and biotic surface colonization.
      ). It remains to be seen whether this gene product is active in Vibrio or other bacteria as we demonstrate for Francisella and whether it plays any role in virulence and/or environmental persistence.
      How does the carboxypeptidase function of REP34 provide a mechanistic basis for the ability of F. tularensis to infect phagocytic host cells? In the absence of specific cellular and molecular mechanistic detail, we can only speculate as to the exact nature of the enzyme's biological role; however, numerous possibilities exist. For instance, REP34 may have a specific host target protein that, when cleaved, would interrupt lysis signaling pathways or promote phagocytosis of the bacterial invader. Given the structural deviation from conventional funnelins, REP34 may have a more specific target than a general basic C terminus, thus limiting the polypeptide sequence space of the host target. Furthermore, the identification of other potential REP-inducing proteins in F. tularensis suggests a complementary role among the various proteins because rREP34 alone is insufficient to complement the REP phenotype to a level approaching wild-type NOV (Fig. 1A). Future efforts to elucidate the host cell targets of the peptidase REP34 and other REP proteins may further shed light on their functions during F. tularensis infection of phagocytic cells.

      Acknowledgments

      We thank D. Finley and the Francis Laboratory at the University of California, Berkeley, for assistance with MALDI-TOF; D. Cascio and M. Sawaya of the UCLA-Department of Energy Institute for Genomics and Proteomics for assistance with crystallographic software; M. Soltis and the 12-2 beamline staff at the Stanford Synchrotron Radiation Laboratory; C. Ogata and the GM/CA 23-ID beamline staff; and M. Capel, K. Rajashankar, N. Sukumar, J. Schuermann, I. Kourinov, F. Murphy, S. Banerjee, K. Perry and D. Neau of the Northeastern Collaborative Access Team beamline 24-ID at the Advanced Photon Source.

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