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Principal Investigator: Karen N. Allen
Organization: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
Fiscal Year: 2022
Award: $437,559
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. An important
mechanism for the assembly of these structures 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 (NDP) activated donor to a PrenP acceptor, yielding
a membrane-bound polyprenol diphosphosugar. Our studies focus on a PGT superfamily with a monotopic
membrane topology (monoPGTs) for which, until our recent studies, there has been only limited structural
and mechanistic information. These enzymes differ from the well-known polytopic PGTs (polyPGTs), which
bear many membrane-spanning sequences. Biochemical studies and the structure of Campylobacter
concisus PglC, show that the monoPGTs include a reentrant membrane helix (RMH) that penetrates only
one leaflet of the bilayer, then re-emerges. This program will pursue synergistic biochemical, bioinformatic,
structural and chemical biology studies of the monoPGTs. In Aim 1 structures will be determined via X-ray
crystallography with detergent-solubilized protein and, in a membrane environment, by solubilization into
lipid nanoparticles and crystallization in the lipidic cubic phase. Cryo-EM in lipid nanoparticles will also be
pursued for members of optimal size. Together with substrate and inhibitor liganded structures and activity
analysis, we will elucidate the specificity determinants of newly-identified monoPGTs and provide
information on their function in the glycoconjugate biosynthetic pathways of various pathogens. In Aim 2,
the model that binding of the UDP-sugar substrate triggers the movement of a soluble loop to complete
substrate-binding determinants and close the active site for catalysis, will be tested using cross-linking and
fluorescence-based approaches in detergent-solubilized and model membrane environments. To provide
complementary insight into the binding of the membrane-resident PrenP substrate, the RMH sequences
will be analyzed via informatics. This information will be used to develop hidden Markov models to identify
RMH segments within the monotopic PGT superfamily and used to predict RMHs in unrelated proteins
families across the proteome. Aim 3 will develop nucleoside analogs that will serve as inhibitors and
activity-based protein profiling probes of the monotopic PGT superfamily. This analysis will define the
contribution of ligand moieties to binding and identify new PGTs and their significance in bacterial
metabolism and host infection. Ultimately, the identified proteins can act as targets for the development of
new antibacterial and antivirulence agents. Overall, this in-depth study of the structures and binding
landscape of the monoPGT superfamily and design of biological probes will establish the fundamental
knowledge and tools needed for validating and intervening in the action of potential therapeutic targets.
Terms: <Active Sites><Anabolism><Anti-Bacterial Agents><Antibacterial Agents><Antigenic Determinants><Bacteria><Bears><Behavior><Binding><Binding Determinants><Bio-Informatics><Biochemical><Bioinformatics><Biologic Models><Biological><Biological Models><Biology><C jejuni><C. jejuni><Campylobacter><Campylobacter jejuni><Catalysis><Cell Body><Cell membrane><Cells><Cellular Membrane><Chemicals><Complex><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><Cysteine><Cytoplasmic Membrane><D-Ribose><Dependence><Detergents><Development><Electron Cryomicroscopy><Environment><Enzyme Gene><Enzymes><Epitopes><Face><Family><Fluorescence><Foundations><Glycans><Glycoconjugates><Half-Cystine><Human><Infection><Informatics><Integral Membrane Protein><Intrinsic Membrane Protein><Knowledge><L-Cysteine><Label><Lead><Ligands><Lipids><Macromolecular Structure><Membrane><Model System><Modeling><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Interaction><Molecular Stereochemistry><Molecular Structure><Movement><Pathogenesis><Pathway interactions><Pb element><Phase><Phosphates><Plasma Membrane><Play><Polysaccharides><Process><Protein Family><Proteins><Proteome><Ribose><Roentgen Rays><Role><Scaffolding Protein><Single Crystal Diffraction><Solid><Specificity><Structure><Substrate Specificity><Testing><Therapeutic><Transferase><Transferase Gene><Transmembrane Protein><Transmembrane Protein Gene><UDP Sugars><Urd><Uridine><Uridine Diphosphate Sugars><Ursidae><Ursidae Family><Virulence><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><activity-based protein profiling><anti-bacterial><antibacterial><bacteria metabolism><bacteria pathogen><bacterial metabolism><bacterial pathogen><base><bear><biologic><biophysical analysis><biophysical studies><biosynthesis><body movement><cell envelope><conformation><conformational state><conformer><cross-link><crosslink><cryo-EM><cryoEM><defined contribution><design><designing><developmental><faces><facial><flexibility><flexible><heavy metal Pb><heavy metal lead><human pathogen><inhibitor><inorganic phosphate><insight><lipid based nanoparticle><lipid nanoparticle><markov model><member><membrane model><membrane structure><molecular dynamics><nucleoside analog><nucleoside diphosphate><pathogen><pathogenic bacteria><pathway><plasmalemma><programs><protein folding><scaffold><scaffolding><small molecule><social role><structural biology><sugar><sugar nucleotide><symbiont><therapeutic target><tool>