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Principal Investigator: Karen N. Allen
Organization: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Fiscal Year: 2024
Award: $453,520
Funding agency: National Institute of General Medical Sciences
Complex glycoconjugates play a pivotal role in bacterial survival, colonization, and virulence, and
contribute to the interactions between symbiotic and pathogenic bacteria and their human hosts. Assembly
of these macromolecules is initiated on the cytoplasmic face of cell membranes, catalyzed by polyprenol
phosphate (PrenP) phosphoglycosyl transferases (PGTs). PGTs transfer a C1’-phosphosugar from a
soluble nucleoside diphosphate-sugar to a PrenP acceptor, yielding a membrane-bound polyprenol
diphosphosugar. Our studies focus on the exclusively prokaryotic PGT superfamily with a monotopic
membrane topology (monoPGTs). Our work has previously led to the mechanistic and structural
characterization of the monoPGTs, revealing a unique reentrant membrane helix supporting the structure
of the active-site residues and substrate-binding determinants. Identification of this core fold has enabled
bioinformatic analysis of sequences from diverse bacteria where the gene encoding the PGT enables
identification of the “signature step” in a dedicated set of genes that, together, describe the glycan of the
glycoconjugate product. The proposed studies will investigate the structures and binding landscapes of
the monoPGT superfamily, and the design of biological probes will establish the fundamental knowledge
and tools needed for validating and intervening in the action of potential therapeutic targets. In Aim 1,
sequence similarity networks will guide the choice of candidates for X-ray crystallographic analysis that
will be determined with detergent-solubilized protein in the small (Sm) monoPGTs, which encodes the core
fold without elaboration. Substrate and inhibitor-liganded structures and activity analysis will elucidate the
determinants of substrate specificity. Genome neighborhood networks will inform on the presence of genes
in the operon that catalyze the biosynthesis of unusual sugars to be tested as substrates. Aim 2 will address
the pathway regulation and flux assisted by the determination of X-ray and CryoEM structures. Protein-
protein interactions will be analyzed via covariance analysis and elucidation of the structure of bifunctional
(Bi) monoPGTs, fusions of monoPGTs and glycosyltransferases, which will also define membrane
interactions and electrostatics. The binding of nucleotides to the proposed regulatory domain of unknown
function (DUF) present in the large (Lg) monoPGTs and dehydrogenases in the pathway will be tested.
Aim 3 builds on nucleoside analogs from solid-phase synthesis and non-hydrolyzable uridine
bisphosphonate-sugars (UBPs) as probes and inhibitors for the prokaryotic monoPGT superfamily. The
structure of UBPs bound to monoPGT targets in Aim 1 will inform further design and identification of
specificity determinants. Overall, these comprehensive and in-depth studies will provide a detailed
structural and functional understanding of this untapped bacterial enzyme superfamily and knowledge of
their glycoconjugate pathways and cellular function.
Terms: <Active Sites><Anabolism><Anti-Bacterial Agents><Antigenic Determinants><Assay><Bacteria><Base Sequence><Binding><Binding Determinants><Bio-Informatics><Bioassay><Biochemical><Biochemistry><Bioinformatics><Biological><Biological Assay><Biological Chemistry><Biology><Biopolymers><Bisphosphonates><Cell Function><Cell Physiology><Cell Process><Cell membrane><Cellular Function><Cellular Physiology><Cellular Process><Chemicals><Classification><Communities><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><Cytoplasmic Membrane><Dedications><Dehydrogenases><Detergents><Development><Diphosphates><E coli><E. coli><EC 2.4><Electron Cryomicroscopy><Electrostatics><Enzyme Gene><Enzymes><Epitopes><Escherichia coli><Face><Family member><Fingerprint><Genes><Genome><Glycans><Glycoconjugates><Glycoside Transferases><Goals><High Throughput Assay><Human><In Vitro><Knowledge><Libraries><Ligands><Membrane><Modern Man><Molecular Interaction><Neighborhoods><Nucleotide Sequence><Nucleotides><O element><O2 element><Operon><Oxidoreductase><Oxidoreductase Gene><Oxygen><Pathogenesis><Pathway interactions><Phenotype><Phosphates><Plasma Membrane><Play><Polysaccharides><Proteins><Pyrophosphates><Reductases><Regulation><Resolution><Roentgen Rays><Role><S Period><S phase><SEQ-AN><Sequence Alignment><Sequence Analyses><Sequence Analysis><Single Crystal Diffraction><Solid><Specificity><Structure><Subcellular Process><Substrate Specificity><Synthesis Period><Synthesis Phase><Systematics><Testing><Thymidin><Thymidine><Transferase><Transferase Gene><UDP Sugars><Urd><Uridine><Uridine Diphosphate Sugars><Virulence><Work><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><analog><anti-bacterial><bacteria pathogen><bacterial pathogen><biologic><biosynthesis><biphosphonate><bisphosphonate><carbene><cell envelope><computational studies><computer studies><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><developmental><diphosphonate><empowerment><faces><facial><gene function><glycosyltransferase><high throughput screening><in vivo><inhibitor><inorganic phosphate><insight><lipid based nanoparticle><lipid nanoparticle><macromolecule><member><membrane structure><meter><methylene><nucleic acid sequence><nucleoside analog><nucleoside diphosphate><parallel computation><parallel computer><parallel computing><pathogenic bacteria><pathway><plasmalemma><prevent><preventing><protein folding><protein protein interaction><resolutions><sequencing alignment><small molecule><social role><structural biology><sugar><sugar nucleotide><symbiont><synergism><therapeutic target><tool>