Browsing by Subject "Peptidoglycan"
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Item Open Access FtsZ Protofilament Curvature Is the Opposite of Tubulin Rings.(Biochemistry, 2016-07) Housman, Max; Milam, Sara L; Moore, Desmond A; Osawa, Masaki; Erickson, Harold PFtsZ protofilaments (pfs) form the bacterial cytokinetic Z ring. Previous work suggested that a conformational change from straight to curved pfs generated the constriction force. In the simplest model, the C-terminal membrane tether is on the outside of the curved pf, facing the membrane. Tubulin, a homologue of FtsZ, also forms pfs with a curved conformation. However, it is well-established that tubulin rings have the C terminus on the inside of the ring. Could FtsZ and tubulin rings have the opposite curvature? In this study, we explored the FtsZ curvature direction by fusing large protein tags to the FtsZ termini. Thin section electron microscopy showed that the C-terminal tag was on the outside, consistent with the bending pf model. This has interesting implications for the evolution of tubulin. Tubulin likely began with the curvature of FtsZ, but evolution managed to reverse direction to produce outward-curving rings, which are useful for pulling chromosomes.Item Open Access Insights into the Structure and Mechanism of Anhydromuramic Acid Kinase (AnmK): A Novel Peptidoglycan Recycling Enzyme with Dual Hydrolase and Kinase Functionality(2011) Allen, Catherine LeighBacteria recycle pre-existing peptidoglycan in order to minimize the de novo synthesis of peptidoglycan precursors. The recycling pathway is under study for its chemotherapeutic target potential. Anhydromuramic acid kinase (AnmK) is part of this recycling pathway and catalyzes the dual hydrolysis/phosphorylation of anhMurNAc to MurNAc-6-P. This enzyme has been discovered and introduced, but only minimally characterized. Therefore, the overarching goal of this work was to clone, express and purify AnmK to homogeneity; perform further kinetic characterization; solve the open, closed and transition state mimic-bound conformations of AnmK by x-ray crystallography; and develop a putative mechanism based on the accumulated research findings and 18O-labeling studies.
The anmK gene was successfully cloned as a hexahistidine fusion protein and overexpression was optimized. After exhaustive trials, a final purification scheme was designed to yield homogeneous AnmK in three chromatographic steps and in less than 36 hours. Additionally, the synthesis of both anhMurNAc and a pseudosubstrate (anhGlcNAc) were carried out in 35% and 77% overall yield, respectively. The synthesis of these compounds allowed for both kinetic characterization and structural studies.
To this end, the structure of de novo AnmK was solved using SAD and high-resolution (1.9 Å) data. Also, an ATP analog (ANP) and anhMurNAc substrate-bound, closed conformation structure (1.95 Å) was solved. These structures elucidated an 11° domain closure of the enzyme upon substrate binding and also revealed the active site geometry to be used to determine potential molecular determinants of specificity.
Insights into the kinetic mechanism of AnmK were then gathered using multiple techniques. First, the structure of AnmK (2.5 Å) was solved the with a known transition state analog, the MgADP-vanadate complex. Following this structure, which sheds light on the potential importance of a residue other than the catalytic base (Asp187), isotopic labeling was performed with H218O. Using NMR and MS, the regiochemical selectivity of AnmK hydrolysis to impart the solvent derived oxygen at C1 was established. Additionally, this was carried out with stereochemical preference to create the α-anomer of the carbohydrate product. This regiochemistry and stereospecificity drove the design of our putative concomitant hydrolysis/phosphorylation mechanism but we are not able to rule out the formation of a transient phosphoenzyme intermediate.
This research can be applied to the immediate goal of understanding the function of a single, novel enzyme with unique chemistry and the clarification of the AnmK mechanism will facilitate future investigation into enzymes with dual hydrolase/kinase functionality. Furthermore, this research contributes to understanding of the complex bacterial peptidoglycan layer in order to harness new ideas for developing antibiotics.
Item Open Access Modulation of bacterial outer membrane vesicle production by envelope structure and content.(BMC Microbiol, 2014-12-21) Schwechheimer, Carmen; Kulp, Adam; Kuehn, Meta JBACKGROUND: Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where outer membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of outer membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. RESULTS: In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in "periplasmic pressure" resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. CONCLUSIONS: From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.Item Open Access NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli.(Microbiologyopen, 2015-06) Schwechheimer, Carmen; Rodriguez, Daniel L; Kuehn, Meta JOuter membrane vesicles (OMVs) are ubiquitously secreted from the outer membrane (OM) of Gram-negative bacteria. These heterogeneous structures are composed of OM filled with periplasmic content from the site of budding. By analyzing mutants that have vesicle production phenotypes, we can gain insight into the mechanism of OMV budding in wild-type cells, which has thus far remained elusive. In this study, we present data demonstrating that the hypervesiculation phenotype of the nlpI deletion mutant of Escherichia coli correlates with changes in peptidoglycan (PG) dynamics. Our data indicate that in stationary phase cultures the nlpI mutant exhibits increased PG synthesis that is dependent on spr, consistent with a model in which NlpI controls the activity of the PG endopeptidase Spr. In log phase, the nlpI mutation was suppressed by a dacB mutation, suggesting that NlpI regulates penicillin-binding protein 4 (PBP4) during exponential growth. The data support a model in which NlpI negatively regulates PBP4 activity during log phase, and Spr activity during stationary phase, and that in the absence of NlpI, the cell survives by increasing PG synthesis. Further, the nlpI mutant exhibited a significant decrease in covalent outer membrane (OM-PG) envelope stabilizing cross-links, consistent with its high level of OMV production. Based on these results, we propose that one mechanism wild-type Gram-negative bacteria can use to modulate vesiculation is by altering PG-OM cross-linking via localized modulation of PG degradation and synthesis.Item Open Access Outer Membrane Vesicle Production in Escherichia coli Relieves Envelope Stress and is Modulated by Changes in Peptidoglycan(2014) Schwechheimer, CarmenBacterial outer membrane vesicles (OMVs) are spherical buds of the outer membrane (OM) containing periplasmic lumenal components. OMVs have been demonstrated to play a critical part in the transmission of virulence factors, immunologically active compounds, and bacterial survival, however vesiculation also appears to be a ubiquitous physiological process for Gram-negative bacteria. Despite their characterized biological roles, especially for pathogens, very little is known about their importance for the originating organism as well as regulation and mechanism of production. Only when we have established their biogenesis can we fully uncover their roles in pathogenesis and bacterial physiology. The overall goal of this research was to characterize bacterial mutants which display altered vesiculation phenotypes using genetic and biochemical techniques, and thereby begin to elucidate the mechanism of vesicle production and regulation. One part of this work elucidated a synthetic genetic growth defect for a strain with reduced OMV production (ΔnlpA, inner membrane lipoprotein with a minor role in methionine transport) and envelope stress (ΔdegP, dual function periplasmic chaperone/ protease responsible for managing proteinaceous waste). This research showed that the growth defect of ΔnlpAΔdegP correlated with reduced OMV production with respect to the hyprevesiculator ΔdegP and the accumulation of protein in the periplasm and DegP substrates in the lumen of OMVs. We further demonstrated that OMVs do not solely act as a stress response pathway to rid the periplasm of otherwise damaging misfolded protein but also of accumulated peptidoglycan (PG) fragments and lipopolysaccharide (LPS), elucidating OMVs as a general stress response pathway critical for bacterial well-being. The second part of this work, focused on the role of PG structure, turnover and covalent crosslinks to the OM in vesiculation. We established a direct link between PG degradation and vesiculation: Mutations in the OM lipoprotein nlpI had been previously established as a very strong hypervesiculation phenotype. In the literature NlpI had been associated with another OM lipoprotein, Spr that was recently identified as a PG hydrolase. The data presented here suggest that NlpI acts as a negative regulator of Spr and that the ΔnlpI hypervesiculation phenotype is a result of rampantly degraded PG by Spr. Additionally, we found that changes in PG structure and turnover correlate with altered vesiculation levels, as well as non-canonical D-amino acids, which are secreted by numerous bacteria on the onset of stationary phase, being a natural factor to increase OMV production. Furthermore, we discovered an inverse relationship between the concentration of Lpp-mediated, covalent crosslinks and the level of OMV production under conditions of modulated PG metabolism and structure. In contrast, situations that lead to periplasmic accumulation (protein, PG fragments, and LPS) and consequent hypervesiculation the overall OM-PG crosslink concentration appears to be unchanged. Form this work, we conclude that multiple pathways lead to OMV production: Lpp concentration-dependent and bulk driven, Lpp concentration-independent.
Item Embargo Structural Studies of Bacterial Cell Wall Synthesis and Remodeling(2023) Hao, AiliBacterial peptidoglycan (PG) cell wall is an essential structural element, providing structural integrity and protection. The essentiality and uniqueness of PG make it an ideal target for antibiotics as over 50% of all antibiotics in clinical use target the PG biosynthesis pathway. PG synthesis involves precursor synthesis in the cytoplasm, transport across the membrane, and final assembly in the periplasmic space to form functional peptidoglycan. PG is also a dynamic structure, as it requires synthesis and degradation in a highly coordinated manner to prevent cell lysis. Despite decades of research, the cellular mechanisms regulating peptidoglycan synthesis and remodeling machineries have remained largely mysterious, particularly at the membrane-bound steps.To understand this essential process, we propose to investigate the structure, mechanism, and inhibition of the proteins and protein complexes involved in the PG pathway during cell division. My dissertation study focuses on key enzymes involved in PG precursor translocation (MraY), transport (MurJ), assembly (FtsBLQWI) and remodeling (FtsEX-EnvC). We employed single-particle cryo-electron microscopy (cryo- EM) to study the inhibition of MraY and MurJ, and to elucidate the regulatory mechanisms of protein complexes FtsBLQWI and FtsEX-EnvC from structural biology standpoint. We utilized in vitro biochemical assays to characterize the biochemical function of these protein complexes. The first part of the work studies PG precursor translocase and transport proteins MraY and MurJ, respectively. MraY catalyzes the reaction that anchors precursor on the membrane, while MurJ then flips the precursor across membrane to the periplasm. MraY has been a target for many naturally occurring nucleoside inhibitors. Rational design and synthesis of natural inhibitor analogs have been a strategy for discovering new and better antibiotics. In collaboration with Dr. Satoshi Ichikawa, we designed and constructed libraries of natural MraY inhibitor analogs. We identified promising MraY inhibitors from these libraries and several showed potent inhibitory and antibacterial activity. We reported two high-resolution cryo-EM structures of MraY bound to the new inhibitor analogs. The structures reveal a novel mode of inhibition, which provides a promising direction in antibiotics design. MurJ has been reported to be inhibited by chemically diverse compounds, but the mechanisms of MraY inhibition by these compounds have been elusive. Understanding MurJ inhibition is important because MurJ is considered a promising new drug target for antibiotic development. We attempted to elucidate the inhibition mechanism of MurJ by capturing MurJ with inhibitor(s) bound using X-ray crystallography. However, despite numerous attempts, the structural characterization of MurJ with inhibitors remained challenging. To overcome the challenges, we generated initial cryo-EM data and proposed strategies for future structural studies of MurJ using cryo-EM. The second part explores the core PG synthesis enzyme complex FtsWI and the accessory regulatory complex FtsBLQ, aiming to understand the interaction and regulation of these essential five proteins during cell division. The molecular regulation within the complex is unknown due to lack of structural information. We attempted to obtain the structure of FtsBLQWI using cryo-EM to elucidate the regulatory mechanism. I co-expressed and obtained homogeneous protein complex from an overexpression system. I developed a functional assay that circumvented the problem of substrate inaccessibility by in situ synthesis of the substrate and demonstrated that the purified complex had robust activity. While still in progress, we identified biochemical condition that yielded promising results for 3D reconstruction, laying the groundwork for future structural investigation on FtsBLQWI. The final part delves into the mechanism of FtsEX-EnvC complex function, which regulates the degradation of PG during cell division. FtsEX-EnvC complex controls PG remodeling by regulating the enzymes that hydrolyze PG. However, the molecular details of how this complex work is unknown. We obtained the cryo-EM structure of the full-length FtsEX-EnvC complex in different nucleotide states, revealing an unexpected conformation in the presence of ADP and providing a novel model on the activation mechanism through spatial regulation. Overall, the study provides structural, inhibitory, and mechanistic insights into several components of the PG pathway. Peptidoglycan synthesis and remodeling are not only fundamental processes in bacterial biology, but also a rich source of antibiotic drug targets. In light of rapidly emerging antimicrobial resistance, understanding the pathways governing PG synthesis and remodeling is crucial for discovering novel targets to develop new inhibitors.
Item Open Access Structural Studies on the Lipid Flippase MurJ(2018) Kuk, Alvin Chun YinThe biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates membrane transport of oligosaccharide precursors from their cytoplasmic site of synthesis to their site of assembly outside the cytoplasm. To address this problem, cells utilize transporters such as those of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily to flip lipid-linked oligosaccharides across the cytoplasmic membrane. The MOP superfamily member MurJ has been shown to be the flippase that transports the lipid-linked peptidoglycan precursor lipid II, but the lack of structural information has limited our mechanistic understanding of the MurJ transport cycle. We determined the first crystal structure of MurJ (MurJTA from Thermosipho africanus) to 2.0-Å resolution, which assumed an inward-facing conformation unlike all other outward-facing structures of MOP transporters. Our structural and mutagenesis studies provide insight into a putative model of lipid II binding and an alternating-access mechanism of transport.