Structural and functional characterization of phosphoglycosyl transferases from human pathogens

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Principal Investigator: Gregory J Dodge
Organization: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Fiscal Year: 2019
Award: $61,226
Funding agency: National Institute of General Medical Sciences

Abstract:
 This project aims to expand the mechanistic understanding of the initial membrane-associated steps of
bacterial glycoconjugate biosynthesis. Membrane protein structures are critically underrepresented in the
protein data bank (PDB), and remain difficult targets for purification, characterization and mechanistic analysis.
The products of these biosynthetic pathways are required for both bacterial viability and virulence and are
attractive targets for antimicrobial design. Examples include capsular polysaccharide (CPS), cell wall teichoic
acid, lipopolysaccharide (LPS) and N- and O-linked glycoproteins.
The first membrane-bound step of glycoconjugate biosynthesis is catalyzed by phosphoglycosyl transferases
(PGTs). This class of enzymes transfers a sugar from an NDP-sugar onto an undecaprenyl phosphate lipid
anchor. Different PGTs exhibit different substrate selectivity, the molecular determinants of which remain
unknown. The first structure of a PGT, PglC from Campylobacter concisus was solved recently, but crystallized
in a conformation in which the active site is open and unliganded.
Both Styrene maleic acid copolymer (SMALP) and traditional detergents will be used to solubilize and purify
PGTs of differing substrate selectivity. Chemoenzymatic synthesis will be used to generate UDP-sugar
substrates for PGTs such as UDP-diNAcBac and UDP-fucose. A rapid, luminescence-based assay will be
used to characterize solubilized targets and synthesized substrates. Lipid cubic phase (LCP) methods will
facilitate crystallization of SMALP solubilized targets that have never left a lipid bilayer. Synthetic substrates
will be utilized for soaking or cocrystallization experiments. These experiments will broaden our understanding
of PGT structure-function relationships. Data will also inform efforts to develop inhibitors, as PGTs remain an
underexplored area for the development of antimicrobial and antivirulence agents.

Terms: <Active Sites><Address><Age><Anabolism><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Area><Assay><Bioassay><Biochemical><Biologic Assays><Biological><Biological Assay><Biological Function><Biological Process><C jejuni><C. jejuni><C.jejuni><Campylobacter><Campylobacter jejuni><Cell Wall><Cell surface><Choline Chloride Dihydrogen Phosphate><Choline Phosphate><Choline Phosphate Chloride><Cocrystallization><Cocrystallography><Crystallization><Data><Data Banks><Databanks><Deoxygalactose><Detergents><Development><Diffusion><Domestic Rabbit><E coli><E. coli><Environment><Enzyme Gene><Enzymes><Escherichia coli><Exhibits><Family member><Fucose><Future><Glycans><Glycoconjugates><Glycoproteins><In Vitro><Integral Membrane Protein><Intrinsic Membrane Protein><Left><Libraries><Link><Lipid Bilayers><Lipids><Lipopolysaccharides><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Methods><Miscellaneous Antibiotic><Molecular><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><Nucleosides><Oryctolagus cuniculus><Pathway interactions><Phase><Phosphates><Phosphatides><Phosphocholine><Phospholipids><Phosphorylcholine><Phosphorylcholine Chloride><Play><Polymers><Polysaccharides><Proteins><Rabbits><Rabbits Mammals><Reporting><Resistance to antibiotics><Resistant to antibiotics><S aureus><S. aureus><S.aureus><Single Crystal Diffraction><Specificity><Staph aureus><Staphylococcus aureus><Structural Protein><Structure><Structure-Activity Relationship><Styrenes><Substrate Interaction><Substrate Specificity><Sugar Phosphates><Surface Proteins><Techniques><Technology><Teichoic Acids><Transferase><Transferase Gene><Transmembrane Protein><Transmembrane Protein Gene><UDP Sugars><Uridine Diphosphate Sugars><Virulence><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><ages><alpha-Fucose><anti-microbial><antibiotic drug resistance><antibiotic resistant><antimicrobial><bacterial pathogen><base><biosynthesis><chemical structure function><colanic acid><conformation><conformational state><copolymer><data repository><data warehouse><design><designing><developmental><enzyme substrate complex><experiment><experimental research><experimental study><extracellular><human pathogen><immunogenicity><in vivo><inhibitor><inhibitor/antagonist><inorganic phosphate><insight><lipid bilayer membrane><lipid-linked oligosaccharides><lipooligosaccharide><luminescence><macromolecule><maleic acid><membrane structure><nano particle><nano-sized particle><nanoparticle><nanosized particle><novel><pathogen><pathogenic bacteria><pathway><prevent><preventing><protein purification><protein structure><screening><structure function relationship><sugar><vapor><vector>