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At least 991 records · Page 55
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1

Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the “rolling hairpin” replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Kinetic Growth of Multicomponent Microcompartment Shells

An important goal of systems and synthetic biology is to produce high value chemical species in large quantities. Microcompartments, which are protein nanoshells encapsulating catalytic enzyme cargo, could potentially function as tunable nanobioreactors inside and outside cells to generate these high value species. Modifying the morphology of microcompartments through genetic engineering of shell proteins is one viable strategy to tune cofactor and metabolite access to encapsulated enzymes. However, this is a difficult task without understanding how changing interactions between the many different types of shell proteins and enzymes affect microcompartment assembly and shape. Here, we use multiscale molecular dynamics and experimental data to describe assembly pathways available to microcompartments composed of multiple types of shell proteins with varied interactions. As the average interaction between the enzyme cargo and the multiple types of shell proteins is weakened, the shell assembly pathway transitions from (i) nucleating on the enzyme cargo to (ii) nucleating in the bulk and then binding the cargo as it grows to (iii) an empty shell. Atomistic simulations and experiments using the 1,2-propanediol utilization microcompartment system demonstrate that shell protein interactions are highly varied and consistent with our multicomponent, coarse-grained model. Furthermore, our results suggest that intrinsic bending angles control the size of these microcompartments. Altogether, our simulations and experiments provide guidance to control microcomparmtent size and assembly by modulating the interactions between shell proteins.

defects↗
DOE OSTI2021

A structured RNA motif locks Argonaute2:miR-122 onto the 5’ end of the HCV genome

microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5’ end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease

ABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

MarK, a Novosphingobium aromaticivorans kinase required for catabolism of multiple aromatic monomers

The aromatic compounds used in a variety of industrial products are currently obtained from nonrenewable petroleum sources. Alternatively, the plant polymer lignin is an abundant renewable source of aromatics, and its depolymerization generates a variety of products that can include acetovanillone, a vanillin derivative containing an acetyl side chain. The Alphaproteobacterium Novosphingobium aromaticivorans DSM12444 can metabolize several chemically modified aromatics in deconstructed lignin, but not acetovanillone. In this work, adaptive laboratory evolution identified a single amino acid change in the previously uncharacterized gene product Saro_1862 that is necessary and sufficient for N. aromaticivorans growth with acetovanillone as a sole growth substrate, as well as other aromatic monomers not metabolized by wild-type cells. We show that a glutamate (E) to lysine (K) substitution at amino acid residue 16 of Saro_1862 results in a ~1600-fold increase in the rate of ATP-dependent acetovanillone phosphorylation. We also find that recombinant Saro_1862 E16K phosphorylates several other aromatic compounds in vitro , defining the first reported catalytic activity for the widespread UPF0261 protein domain contained in Saro_1862. Thus, we propose naming Saro_1862 MarK, for multiple aromatic kinase. A 1.57 Å crystal structure of MarK E16K predicts that the E16K substitution lies in a potential ATP binding site, suggesting how this amino acid change increased catalytic activity. A search for homologs of MarK and other proteins required for acetovanillone degradation predicts that this pathway for aromatic metabolism exists throughout the bacterial phylogeny.

Novosphingobium↗
DOE OSTI2022

Decoding the protein–ligand interactions using parallel graph neural networks

Abstract Protein–ligand interactions (PLIs) are essential for biochemical functionality and their identification is crucial for estimating biophysical properties for rational therapeutic design. Currently, experimental characterization of these properties is the most accurate method, however, this is very time-consuming and labor-intensive. A number of computational methods have been developed in this context but most of the existing PLI prediction heavily depends on 2D protein sequence data. Here, we present a novel parallel graph neural network (GNN) to integrate knowledge representation and reasoning for PLI prediction to perform deep learning guided by expert knowledge and informed by 3D structural data. We develop two distinct GNN architectures: $$\hbox {GNN}_{\mathrm{F}}$$ GNN F is the base implementation that employs distinct featurization to enhance domain-awareness, while $$\hbox {GNN}_{\mathrm{P}}$$ GNN P is a novel implementation that can predict with no prior knowledge of the intermolecular interactions. The comprehensive evaluation demonstrated that GNN can successfully capture the binary interactions between ligand and protein’s 3D structure with 0.979 test accuracy for $$\hbox {GNN}_{\mathrm{F}}$$ GNN F and 0.958 for $$\hbox {GNN}_{\mathrm{P}}$$ GNN P for predicting activity of a protein–ligand complex. These models are further adapted for regression tasks to predict experimental binding affinities and $$\hbox {pIC}_{\mathrm{50}}$$ pIC 50 crucial for compound’s potency and efficacy. We achieve a Pearson correlation coefficient of 0.66 and 0.65 on experimental affinity and 0.50 and 0.51 on $$\hbox {pIC}_{\mathrm{50}}$$ pIC 50 with $$\hbox {GNN}_{\mathrm{F}}$$ GNN F and $$\hbox {GNN}_{\mathrm{P}}$$ GNN P , respectively, outperforming similar 2D sequence based models. Our method can serve as an interpretable and explainable artificial intelligence (AI) tool for predicted activity, potency, and biophysical properties of lead candidates. To this end, we show the utility of $$\hbox {GNN}_{\mathrm{P}}$$ GNN P on SARS-Cov-2 protein targets by screening a large compound library and comparing the prediction with the experimentally measured data.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Using diverse potentials and scoring functions for the development of improved machine-learned models for protein–ligand affinity and docking pose prediction

The advent of computational drug discovery holds the promise of significantly reducing the effort of experimentalists, along with monetary cost. More generally, predicting the binding of small organic molecules to biological macromolecules has far-reaching implications for a range of problems, including metabolomics. However, problems such as predicting the bound structure of a protein–ligand complex along with its affinity have proven to be an enormous challenge. In recent years, machine learning-based methods have proven to be more accurate than older methods, many based on simple linear regression. Nonetheless, there remains room for improvement, as these methods are often trained on a small set of features, with a single functional form for any given physical effect, and often with little mention of the rationale behind choosing one functional form over another. Moreover, it is not entirely clear why one machine learning method is favored over another. Here, we endeavor to undertake a comprehensive effort towards developing high-accuracy, machine-learned scoring functions, systematically investigating the effects of machine learning method and choice of features, and, when possible, providing insights into the relevant physics using methods that assess feature importance. Here, we show synergism among disparate features, yielding adjusted R 2 with experimental binding affinities of up to 0.871 on an independent test set and enrichment for native bound structures of up to 0.913. When purely physical terms that model enthalpic and entropic effects are used in the training, we use feature importance assessments to probe the relevant physics and hopefully guide future investigators working on this and other computational chemistry problems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Osteocalcin binds to a GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A, a member of the Family C G-protein coupled receptors, regulates energy metabolism and sex hormone production and is activated by diverse ligands, including cations, L-amino acids, the osteocalcin (Ocn) peptide and the steroid hormone testosterone. We sought a structural framework for the ability of multiple distinct classes of ligands to active GPRC6A. We created a structural model of GPRC6A using Alphafold2. Using this model we explored a putative orthosteric ligand binding site in the bilobed Venus fly trap (VFT) domain of GPRC6A and two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7 transmembrane (7TM) domain. We provide evidence that Ocn peptides act as a PAM for GPRC6A by binding to a site in the VFT that is distinct from the orthosteric site for calcium and L-amino acids. In agreement with this prediction, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the computationally predicted Ocn binding site, K352E and H355P, prevent Ocn activation of GPRC6A. These observations explain how dissimilar ligands activate GPRC6A and set the stage to develop novel molecules to activate and inhibit this previously poorly understood receptor.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Small molecule microarray identifies inhibitors of tyrosyl-DNA phosphodiesterase 1 that simultaneously access the catalytic pocket and two substrate binding sites

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a member of the phospholipase D family of enzymes, which catalyzes the removal of both 3'- and 5'-DNA phosphodiester adducts. Importantly, it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of human TOP1 and the 3'-phosphate of its DNA substrate. Blocking TDP1 function is an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. Previously, we reported the use of an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. We now report examining a 21 000-member library of drug-like Small Molecules in Microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke–Blackburn–Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. Although our original fragment screen differed significantly from the current microarray protocol, both methods identified ligand–protein interactions containing highly similar elements. Importantly inhibitors identified through the SMM approach show competitive inhibition against TDP1 and access the catalytic phosphate-binding pocket, while simultaneously providing extensions into both the substrate DNA and peptide-binding channels. As such, they represent a platform for further elaboration of trivalent ligands, that could serve as a new genre of potent TDP1 inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose–bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification and characterization of the WYL BrxR protein and its gene as separable regulatory elements of a BREX phage restriction system

Bacteriophage exclusion (‘BREX’) phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR (‘BREX Regulator’), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

NEAr Transporter (NEAT) Domains: Unique Surface Displayed Heme Chaperones That Enable Gram-Positive Bacteria to Capture Heme-Iron From Hemoglobin

Iron is an important micronutrient that is required by bacteria to proliferate and to cause disease. Many bacterial pathogens forage iron from human hemoglobin (Hb) during infections, which contains this metal within heme (iron–protoporphyrin IX). Several clinically important pathogenic species within the Firmicutes phylum scavenge heme using surface-displayed or secreted NEAr Transporter (NEAT) domains. In this review, we discuss how these versatile proteins function in the Staphylococcus aureus Iron-regulated surface determinant system that scavenges heme-iron from Hb. S. aureus NEAT domains function as either Hb receptors or as heme-binding chaperones. In vitro studies have shown that heme-binding NEAT domains can rapidly exchange heme amongst one another via transiently forming transfer complexes, leading to the interesting hypothesis that they may form a protein-wire within the peptidoglycan layer through which heme flows from the microbial surface to the membrane. In Hb receptors, recent studies have revealed how dedicated heme- and Hb-binding NEAT domains function synergistically to extract Hb’s heme molecules, and how receptor binding to the Hb-haptoglobin complex may block its clearance by macrophages, prolonging microbial access to Hb’s iron. The functions of NEAT domains in other Gram-positive bacteria are also reviewed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nucleotide- and metalloid-driven conformational changes in the arsenite efflux ATPase ArsA

Arsenite (As III ) is toxic to all organisms due to its ability to tightly bind exposed thiols within cells. An important As III resistance mechanism in prokaryotes involves proteins encoded by thearsoperon. A central component of thearsoperon in many bacteria is the cytoplasmic ATPase, ArsA, which orchestrates a series of nucleotide-dependent handoffs, starting with the capture of As III by the ArsD metallochaperone and culminating in its removal from the cell by the ArsB efflux pump. Although the mechanism of ArsA has been widely studied, the molecular details of how nucleotide hydrolysis modulates these events remain unclear. ArsA is an archetypal member of the intradimeric Walker A (IWA) family of ATPases, implicated in a diversity of complex biological functions. Conformational changes typical of IWA ATPases have been postulated to drive these molecular events but have not been demonstrated. We report cryogenic electron microscopy (cryo-EM) structures of ArsA in MgADP-bound and MgATP-boundopenstates, as well as a distinctclosedMgATP-bound state liganded to As III . X-ray absorption spectroscopy (XAS) confirmed three-coordinate binding of As III to the conserved cysteines at the metalloid-binding site of the closed state. Coupled with biochemical characterization, our cryo-EM structures reveal key conformational changes in the ArsA catalytic cycle consistent with other IWA ATPases and provide the structural basis for allosteric activation of nucleotide hydrolysis by As III . This work establishes how the nucleotide state of ArsA transiently creates a high-affinity binding site that can sequester metalloid within the cell, followed by a nucleotide-driven handoff to ArsB for efflux.

Science & Technology - Other Topics↗
DOE OSTI2020

Quality-Control Mechanism for Telomerase RNA Folding in the Cell

Long non-coding RNAs can often fold into different conformations. Telomerase RNA, an essential component of the telomerase ribonucleoprotein (RNP) enzyme, must fold into a defined structure to fulfill its function with the protein catalytic subunit (TERT) and other accessory factors. However, the mechanism by which the correct folding of telomerase RNA is warranted in a cell is still unknown. Here we show that La-related protein Pof8 specifically recognizes the conserved pseudoknot region of telomerase RNA and instructs the binding of the Lsm2-8 complex to its mature 3' end, thus selectively protecting the correctly folded RNA from exonucleolytic degradation. In the absence of Pof8, TERT assembles with misfolded RNA and produces little telomerase activity. Therefore, Pof8 plays a key role in telomerase RNA folding quality control, ensuring that TERT only assembles with functional telomerase RNA to form active telomerase. Our finding reveals a mechanism for non-coding RNA folding quality control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Potential pathogenicity determinants identified from structural proteomics of SARS-CoV and SARS-CoV-2

Despite SARS-CoV and SARS-CoV-2 being equipped with highly similar protein arsenals, the corresponding zoonoses have spread among humans at extremely different rates. The specific characteristics of these viruses that led to such distinct outcomes remain unclear. Here, we apply proteome-wide comparative structural analysis aiming to identify the unique molecular elements in the SARS-CoV-2 proteome that may explain the differing consequences. By combining protein modeling and molecular dynamics simulations, we suggest non-conservative substitutions in functional regions of the spike glycoprotein (S), nsp1, and nsp3 that are contributing to differences in virulence. Particularly, we explain why the substitutions at the receptor-binding domain of S affect the structure-dynamics behavior in complexes with putative host receptors. Conservation of functional protein regions within the two taxa is also noteworthy. We suggest that the highly conserved main protease, nsp5, of SARS-CoV and SARS-CoV-2 is part of their mechanism of circumventing the host interferon antiviral response. Overall, most substitutions occur on the protein surfaces and may be modulating their antigenic properties and interactions with other macromolecules. Our results imply that the striking difference in the pervasiveness of SARS-CoV-2 and SARS-CoV among humans seems to significantly derive from molecular features that modulate the efficiency of viral particles in entering the host cells and blocking the host immune response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Characterization of alternate encounter assemblies of SARS-CoV-2 main protease

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ~7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ~6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

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

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.

59 BASIC BIOLOGICAL SCIENCES↗