I04-Macromolecular Crystallography
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Matthew
Singer
,
Tung
Dinh
,
Lev
Levintov
,
Arun S.
Annamalai
,
Juan S.
Rey
,
Lorenzo
Briganti
,
Nicola J.
Cook
,
Valerie E.
Pye
,
Ian A.
Taylor
,
Kyungjin
Kim
,
Alan N.
Engelman
,
Baek
Kim
,
Juan R.
Perilla
,
Mamuka
Kvaratskhelia
,
Peter
Cherepanov
Diamond Proposal Number(s):
[13775]
Open Access
Abstract: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target.
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Feb 2023
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Many bacteria of the genus Shewanella are facultative anaerobes able to reduce a broad range of soluble and insoluble substrates, including Fe(III) mineral oxides. Under anoxic conditions, the bacterium Shewanella oneidensis MR-1 uses a porin-cytochrome complex (Mtr) to mediate extracellular electron transfer (EET) across the outer membrane to extracellular substrates. However, it is unclear how EET prevents generating harmful reactive oxygen species (ROS) when exposed to oxic environments. The Mtr complex is expressed under anoxic and oxygen-limited conditions and contains an extracellular MtrC subunit. This has a conserved CX8C motif that inhibits aerobic growth when removed. This inhibition is caused by an increase in ROS that kills the majority of S. oneidensis cells in culture. To better understand this effect, soluble MtrC isoforms with modified CX8C were isolated. These isoforms produced increased concentrations of H2O2 in the presence of flavin mononucleotide (FMN) and greatly increased the affinity between MtrC and FMN. X-ray crystallography revealed that the molecular structure of MtrC isoforms was largely unchanged, while small-angle X-ray scattering suggested that a change in flexibility was responsible for controlling FMN binding. Together, these results reveal that FMN reduction in S. oneidensis MR-1 is controlled by the redox-active disulfide on the cytochrome surface. In the presence of oxygen, the disulfide forms, lowering the affinity for FMN and decreasing the rate of peroxide formation. This cysteine pair consequently allows the cell to respond to changes in oxygen level and survive in a rapidly transitioning environment.
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Jan 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19242]
Open Access
Abstract: The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of ‘omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how “underground metabolism,” facilitated by enzyme substrate promiscuity, “rewires” Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism.
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Nov 2022
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I03-Macromolecular Crystallography
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Yao
Zhou
,
Kaizhou
Yan
,
Qijian
Qin
,
Olawale G.
Raimi
,
Chao
Du
,
Bin
Wang
,
Chukwuemeka Samson
Ahamefule
,
Bartosz
Kowalski
,
Cheng
Jin
,
Daniel M. F.
Van Aalten
,
Wenxia
Fang
Diamond Proposal Number(s):
[26793]
Open Access
Abstract: Aspergillus fumigatus is a devastating opportunistic fungal pathogen causing hundreds of thousands of deaths every year. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that converts glucose-6-phosphate to fructose-6-phosphate, a key precursor of fungal cell wall biosynthesis. Here, we demonstrate that the growth of A. fumigatus is repressed by the deletion of pgi, which can be rescued by glucose and fructose supplementation in a 1:10 ratio. Even under these optimized growth conditions, the Δpgi mutant exhibits severe cell wall defects, retarded development, and attenuated virulence in Caenorhabditis elegans and Galleria mellonella infection models. To facilitate exploitation of A. fumigatus PGI as an antifungal target, we determined its crystal structure, revealing potential avenues for developing inhibitors, which could potentially be used as adjunctive therapy in combination with other systemic antifungals.
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Aug 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate.
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Jun 2022
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I04-Macromolecular Crystallography
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Open Access
Abstract: The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in D. dadantii, the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex in vivo. We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Philip
Hinchliffe
,
Catherine L.
Tooke
,
Christopher R.
Bethel
,
Benlian
Wang
,
Christopher
Arthur
,
Kate J.
Heesom
,
Stuart
Shapiro
,
Daniela M.
Schlatzer
,
Krisztina M.
Papp-Wallace
,
Robert A.
Bonomo
,
James
Spencer
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: β-Lactamases hydrolyze β-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) β-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum β-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a D-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cβ closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of β-lactamase inhibition by these agents.
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May 2022
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Human gut commensal Bacteroidetes rely on multiple transport systems to acquire vitamin B12 and related cobamides for fitness in the gut. In addition to a set of conserved transport proteins, these systems also include a diverse repertoire of additional proteins with unknown function. Here, we report the function and structural characterization of one of these proteins, BtuH, which binds vitamin B12 directly via a C-terminal globular domain that has no known structural homologs. This protein is required for efficient B12 transport and competitive fitness in the gut, demonstrating that members of the heterogeneous suite of accessory proteins encoded in Bacteroides cobamide transport system loci can play key roles in vitamin acquisition.
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Mar 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14744, 19946]
Open Access
Abstract: Transmission of the New World hemorrhagic fever arenaviruses Junín virus (JUNV) and Machupo virus (MACV) to humans is facilitated, in part, by the interaction between the arenavirus GP1 glycoprotein and the human transferrin receptor 1 (hTfR1). We utilize a mouse model of live-attenuated immunization with envelope exchange viruses to isolate neutralizing monoclonal antibodies (NAbs) specific to JUNV GP1 and MACV GP1. Structures of two NAbs, termed JUN1 and MAC1, demonstrate that they neutralize through disruption of hTfR1 recognition. JUN1 utilizes a binding mode common to all characterized infection- and vaccine-elicited JUNV-specific NAbs, which involves mimicking hTfR1 binding through the insertion of a tyrosine into the receptor-binding site. In contrast, MAC1 undergoes a tyrosine-mediated mode of antigen recognition distinct from that used by the reported anti-JUNV NAbs and the only other characterized anti-MACV NAb. These data reveal the varied modes of GP1-specific recognition among New World arenaviruses by the antibody-mediated immune response.
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Mar 2022
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17773]
Open Access
Abstract: During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of a stress signaling network termed the stringent response. RsgA, RbgA, Era, and HflX are four ribosome-associated GTPases (RA-GTPases) that bind to (p)ppGpp in Staphylococcus aureus. These enzymes are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) ribosome-associated states. Entry into the OFF state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly and growth. Here, we determine how (p)ppGpp impacts RA-GTPase-ribosome interactions. We show that RA-GTPases preferentially bind to 5′-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell to shut down ribosome biogenesis during stress. Stopped-flow fluorescence and association assays reveal that when bound to (p)ppGpp, the association of RA-GTPases to ribosomal subunits is destabilized, both in vitro and within bacterial cells. Consistently, structural analysis of the ppGpp-bound RA-GTPase RsgA reveals an OFF-state conformation similar to the GDP-bound state, with the G2/switch I loop adopting a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated ribosome assembly and growth control.
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Nov 2021
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