ABSTRACT
The ability of histone chaperone Anti-silencing factor 1 (Asf1) to direct acetylation of lysine 56 of histone H3 (H3K56ac) represents an important regulatory step in genome replication and DNA repair. In Saccharomyces cerevisiae, Asf1 interacts functionally with a second chaperone, Vps75, and the lysine acetyltransferase (KAT) Rtt109. Both Asf1 and Vps75 can increase the specificity of histone acetylation by Rtt109, but neither alter selectivity. However, changes in acetylation selectivity have been observed in histones extracted from cells, which contain a plethora of post-translational modifications. In the present study, we use a series of singly acetylated histones to test the hypothesis that histone pre-acetylation and histone chaperones function together to drive preferential acetylation of H3K56. We show that pre-acetylated H3K14ac/H4 functions with Asf1 to drive specific acetylation of H3K56 by Rtt109-Vps75. Additionally, we identified an exosite containing an acidic patch in Asf1 and show that mutations to this region alter Asf1-mediated crosstalk that changes Rtt109-Vps75 selectivity. Our proposed mechanism suggests that Gcn5 acetylates H3K14, recruiting remodeler complexes, allowing for the Asf1-H3K14ac/H4 complex to be acetylated at H3K56 by Rtt109-Vps75. This mechanism explains the conflicting biochemical data and the genetic links between Rtt109, Vps75, Gcn5 and Asf1 in the acetylation of H3K56.
Subject(s)
Cell Cycle Proteins/metabolism , Histones/metabolism , Lysine/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Acetylation , Cell Cycle Proteins/genetics , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Molecular Chaperones/genetics , Mutation , Protein Binding , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Substrate SpecificityABSTRACT
Gram-negative bacterial viability is greatly reduced by the disruption of heptose sugar addition during the biosynthesis of lipopolysaccharide (LPS), an important bacterial outer membrane component. Heptosyltransferase I (HepI), a member of the GT-B structural subclass of glycosyltransferases, is therefore an essential enzyme for the biosynthesis of the LPS. The disruption of HepI also increases the susceptibility of bacteria to hydrophobic antibiotics, making HepI a potential target for drug development. In this work, the structural and dynamic properties of the catalytic cycle of HepI are explored. Previously, substrate-induced stabilization of HepI was observed and hypothesized to be assisted by interactions between the substrate and residues located on dynamic loops. Herein, positively charged amino acids were probed to identify binding partners of the negatively charged phosphates and carboxylates of Kdo2-lipid A and its analogues. Mutant enzymes were characterized to explore changes in enzymatic activities and protein stability. Molecular modeling of HepI in the presence and absence of ligands was then performed with the wild type and mutant enzyme to allow determination of the relative change in substrate binding affinity resulting from each mutation. Together, these studies suggest that multiple residues are involved in mediating substrate binding, and a lack of additivity of these effects illustrates the functional redundancy of these binding interactions. The redundancy of residues mediating conformational transitions in HepI illustrates the evolutionary importance of these structural rearrangements for catalysis. This work enhances the understanding of HepI's protein dynamics and mechanism and is a model for improving our understanding of glycosyltransferase enzymes.
Subject(s)
Escherichia coli/enzymology , Glycosyltransferases/chemistry , Glycosyltransferases/metabolism , Glycosyltransferases/genetics , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , Sequence AlignmentABSTRACT
T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5'-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.
Subject(s)
IMP Dehydrogenase/metabolism , T-Lymphocytes/enzymology , Animals , Cells, Cultured , Guanine Nucleotides/metabolism , IMP Dehydrogenase/genetics , Lymphocyte Activation , Male , Mice , Mice, Inbred C57BL , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Spleen/enzymology , Spleen/immunology , Stromal Interaction Molecule 1/genetics , Stromal Interaction Molecule 1/metabolism , T-Lymphocytes/immunology , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Galactosides/pharmacology , Glucosides/pharmacology , Glycosyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Binding Sites , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Escherichia coli Proteins/chemistry , Galactosides/chemical synthesis , Galactosides/chemistry , Glucosides/chemical synthesis , Glucosides/chemistry , Glycosyltransferases/chemistry , Kinetics , Lipid A/analogs & derivatives , Lipid A/chemical synthesis , Lipid A/chemistry , Lipid A/pharmacology , Molecular Docking SimulationABSTRACT
Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-ß-d-manno-heptose to Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Glycosyltransferases/metabolism , Lipid A/metabolism , Models, Molecular , Acylation , Amino Acid Substitution , Apoenzymes/antagonists & inhibitors , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Binding Sites , Biocatalysis , Circular Dichroism , Enzyme Stability , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Glycosyltransferases/antagonists & inhibitors , Glycosyltransferases/chemistry , Glycosyltransferases/genetics , Lipid A/chemistry , Molecular Dynamics Simulation , Mutagenesis, Site-Directed , Mutation , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Solubility , Solvents/chemistry , Spectrometry, Fluorescence , Surface Properties , Tryptophan/chemistryABSTRACT
Bacterial antibiotic resistance is a rapidly expanding problem in the world today. Functionalization of the outer membrane of Gram-negative bacteria provides protection from extracellular antimicrobials, and serves as an innate resistance mechanism. Lipopolysaccharides (LPS) are a major cell-surface component of Gram-negative bacteria that contribute to protecting the bacterium from extracellular threats. LPS is biosynthesized by the sequential addition of sugar moieties by a number of glycosyltransferases (GTs). Heptosyltransferases catalyze the addition of multiple heptose sugars to form the core region of LPS; there are at most four heptosyltransferases found in all Gram-negative bacteria. The most studied of the four is HepI. Cells deficient in HepI display a truncated LPS on their cell surface, causing them to be more susceptible to hydrophobic antibiotics. HepI-IV are all structurally similar members of the GT-B structural family, a class of enzymes that have been found to be highly dynamic. Understanding conformational changes of heptosyltransferases are important to efficiently inhibiting them, but also contributing to the understanding of all GT-B enzymes. Finding new and smarter methods to inhibit bacterial growth is crucial, and the Heptosyltransferases may provide an important model for how to inhibit many GT-B enzymes.
Subject(s)
Glycosyltransferases/metabolism , Lipopolysaccharides/metabolism , Animals , Catalytic Domain , Glycosyltransferases/chemistry , Humans , Lipopolysaccharides/chemistry , Models, Molecular , Sequence Homology , Structure-Activity RelationshipABSTRACT
A clinically relevant inhibitor for Heptosyltransferase I (HepI) has been sought after for many years because of its critical role in the biosynthesis of lipopolysaccharides on bacterial cell surfaces. While many labs have discovered or designed novel small molecule inhibitors, these compounds lacked the bioavailability and potency necessary for therapeutic use. Extensive characterization of the HepI protein has provided valuable insight into the dynamic motions necessary for catalysis that could be targeted for inhibition. Structural inspection of Kdo2-lipid A suggested aminoglycoside antibiotics as potential inhibitors for HepI. Multiple aminoglycosides have been experimentally validated to be first-in-class nanomolar inhibitors of HepI, with the best inhibitor demonstrating a Ki of 600 ± 90 nM. Detailed kinetic analyses were performed to determine the mechanism of inhibition while circular dichroism spectroscopy, intrinsic tryptophan fluorescence, docking, and molecular dynamics simulations were used to corroborate kinetic experimental findings. While aminoglycosides have long been described as potent antibiotics targeting bacterial ribosomes' protein synthesis leading to disruption of the stability of bacterial cell membranes, more recently researchers have shown that they only modestly impact protein production. Our research suggests an alternative and novel mechanism of action of aminoglycosides in the inhibition of HepI, which directly leads to modification of LPS production in vivo. This finding could change our understanding of how aminoglycoside antibiotics function, with interruption of LPS biosynthesis being an additional and important mechanism of aminoglycoside action. Further research to discern the microbiological impact of aminoglycosides on cells is warranted, as inhibition of the ribosome may not be the sole and primary mechanism of action. The inhibition of HepI by aminoglycosides may dramatically alter strategies to modify the structure of aminoglycosides to improve the efficacy in fighting bacterial infections.