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Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates

Award Number: R01GM131627
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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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.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $0 )
2025	2024	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	6	11/19/2024	COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2024 ( Subtotal = $453,520 )
2024	2024	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	6	7/17/2024	COMPETING CONTINUATION	$453,520
2024	2023	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	5	7/16/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $453,520

Issue Date FY: 2023 ( Subtotal = $437,559 )
2023	2023	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	5	7/11/2023	NON-COMPETING CONTINUATION	$437,559
														Subtotal = $437,559

Issue Date FY: 2022 ( Subtotal = $622,662 )
2022	2022	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	3	5/24/2022	SUPPLEMENT FOR EXPANSION	$185,103
2022	2022	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	003	4	7/11/2022	NON-COMPETING CONTINUATION	$437,559
2022	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	002	3	6/9/2022	COMPETING CONTINUATION	$76,497
2022	2020	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	6/9/2022	NON-COMPETING CONTINUATION	-$76,497
														Subtotal = $622,662

Issue Date FY: 2021 ( Subtotal = $451,334 )
2021	2021	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	3	7/7/2021	COMPETING CONTINUATION	$451,334
2021	2020	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	7/22/2021	NON-COMPETING CONTINUATION	$0
														Subtotal = $451,334

Issue Date FY: 2020 ( Subtotal = $318,411 )
2020	2020	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	2	1/29/2020	NON-COMPETING CONTINUATION	$286,569
2020	2020	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	2	3/4/2020	NON-COMPETING CONTINUATION	$31,842
														Subtotal = $318,411

Issue Date FY: 2019 ( Subtotal = $333,750 )
2019	2019	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	001	1	2/14/2019	NEW	$70,428
2019	2019	MASSACHUSETTS INSTITUTE OF TECHNOLOGY	77 MASSACHUSETTS AVE	CAMBRIDGE	MA	02139	MIDDLESEX	USA	Biomedical Research and Research Training	000	1	1/24/2019	NEW	$263,322
														Subtotal = $333,750

Grand Total All Awards = $2,617,236

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Structure and function of the monotopic phosphoglycosyl transferase superfamily: Initiators of biosynthesis of complex bacterial glycoconjugates

Award Number: R01GM131627

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

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