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Structural basis of sigma-regulator recognition by a site-1 protease during cell surface signaling by Gram-negative bacteria.

Award Number: R01GM126207
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/20/2018
PERIOD OF PERFORMANCE END DATE: 02/28/2030

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Structural basis of sigma-regulator recognition by a site-1 protease during cell surface signaling by Gram-negative bacteria. - PROJECT SUMMARY/ABSTRACT Gram-negative bacteria (GNB) play an important role in human health. GNB include species beneficial to humans as well as some of the most dangerous human pathogens, many of which have evolved to produce multi-drug-resistant strains as emphasized in the 2019 Antibiotic Resistance Threats Report from the CDC. This underscores the need to better understand essential cellular processes in GNB in order to develop new, more potent and specific therapeutics. All GNB import critical nutrients from their environment using a set of homologous, yet highly specific, outer membrane TonB-dependent transporters (TBDTs). To import metals, especially iron, GNB secrete high-affinity, metal-chelating compounds called siderophores, and iron-laden siderophores are imported by specific TBDTs. A subset of these TBDTs transcriptionally up-regulate their own expression by a cell surface signaling (CSS) process. The objective of the research outlined in this proposal is to obtain structural and mechanistic information about regulated intramembrane proteolysis of the s-regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, a model CSS system, and compare it to the E. coli ferric citrate transport (Fec) system, which undergoes an autoproteolysis event in the periplasm and could result in a different mechanism. CSS involves a cytoplasmic transcription factor or s-factor, PupI/FecI, which is localized to the inner membrane by binding to the inner membrane s-regulator, PupR/FecR, and an outer membrane TBDT, PupB/FecA. The C-terminal processing protease, Prc, which is also known as a site-1 protease, activates CSS by proteolyzing the s-regulator. The overall goal of this proposal is to understand the structural and biochemical basis of this process. This will be achieved by successfully accomplishing three specific aims: 1) determining if PupB NTSD prevents recognition of the PupR CCSSD by Prc, and to delineate how Prc processes PupR. 2) elucidating the mechanism of Prc-mediated proteolysis of the PupR CCSSD, and 3) evaluating whether FecR CCSSD autoproteolysis affects FecA NTSD:FecR CCSSD binding. These aims will be accomplished using a multidisciplinary approach involving structural biology methods such as X-ray crystallography, CryoEM, and small-angle X-ray scattering; biophysical methods such as mass spectrometry, microscale thermophoresis, and isothermal titration calorimetry; and biochemical and molecular biology techniques such as SDS-PAGE, site directed mutagenesis, and proteolysis assays. Thus, this research will provide critical structural information explaining how a s-regulator is recognized and degraded by a site-1 protease during activation of CSS. Ultimately, this information will be useful in informing new strategies to modulate TBDT expression. This information may be critical to facilitate the development of novel adjuvants for “Trojan Horse” antibiotic delivery or phage therapy.


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: 2026 ( Subtotal = $310,211 )
2026	2026	NORTH DAKOTA STATE UNIVERSITY	1340 ADMINISTRATION AVE	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	000	5	6/23/2026	COMPETING CONTINUATION	$310,211
														Subtotal = $310,211

Issue Date FY: 2025 ( Subtotal = -$90 )
2025	2021	NORTH DAKOTA STATE UNIVERSITY	1340 ADMINISTRATION AVE	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	000	4	4/2/2025	NON-COMPETING CONTINUATION	-$90
														Subtotal = -$90

Issue Date FY: 2023 ( Subtotal = $0 )
2023	2021	NORTH DAKOTA STATE UNIVERSITY	1310 BOLLEY DR	FARGO	ND	58105	CASS	USA	Biomedical Research and Research Training	000	4	9/4/2023	NON-COMPETING CONTINUATION	$0
														Subtotal = $0

Issue Date FY: 2021 ( Subtotal = $489,943 )
2021	2021	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	4	8/26/2021	NON-COMPETING CONTINUATION	$290,000
2021	2021	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	001	4	8/30/2021	SUPPLEMENT FOR EXPANSION	$199,943
														Subtotal = $489,943

Issue Date FY: 2020 ( Subtotal = $290,000 )
2020	2020	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	3	8/17/2020	NON-COMPETING CONTINUATION	$290,000
														Subtotal = $290,000

Issue Date FY: 2019 ( Subtotal = $290,000 )
2019	2019	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	2	8/29/2019	NON-COMPETING CONTINUATION	$290,000
														Subtotal = $290,000

Issue Date FY: 2018 ( Subtotal = $290,000 )
2018	2018	NORTH DAKOTA STATE UNIVERSITY	1919 UNIVERSITY DR N, STE 102	FARGO	ND	58102	CASS	USA	Biomedical Research and Research Training	000	1	9/20/2018	NEW	$290,000
														Subtotal = $290,000

Grand Total All Awards = $1,670,064

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Structural basis of sigma-regulator recognition by a site-1 protease during cell surface signaling by Gram-negative bacteria.

Award Number: R01GM126207

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 09/20/2018

PERIOD OF PERFORMANCE END DATE: 02/28/2030

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