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NMR crystallography: Imaging active site chemistry and protonation states

Award Number: R35GM145369
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: 08/01/2022
PERIOD OF PERFORMANCE END DATE: 07/31/2027

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NMR crystallography: Imaging active site chemistry and protonation states - My group is working to develop NMR-assisted crystallography – the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry – as an atomic-resolution probe of enzyme active sites, capable of defining the position of all atoms, including hydrogens. By locating hydrogen atoms, this technique provides the often critical missing chemical information necessary to link structure and mechanism, as well as providing crucial information for the rational design of therapeutics. The approach is three-fold: X-ray crystallography is used to provide a coarse structural framework upon which chemically-detailed models of the active site are built using computational chemistry, and various active site chemistries explored; these models can be quantitatively distinguished by comparing their predicted NMR chemical shifts with the results from solid-state NMR experiments. Provided a sufficient number of chemical shift restraints are measured within the active site, NMR-assisted crystallography can uniquely identify the structure. The targeted systems include pyridoxal-5’-phosphate (PLP)-dependent enzymes, which have been implicated in numerous health conditions and as targets for treating diseases, and the β-Lactamases, which mediate antibiotic resistance to β-lactam antibiotics. The family of PLP-dependent enzymes are involved in the metabolism of amino acids and other amine- containing biomolecules. This single cofactor can participate in a diverse array of chemical transformations, including racemization, transamination, α/β-decarboxylation, and α/β/γ- elimination and substitution. Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of this proposal. To accomplish this understanding, NMR-assisted crystallography is employed to characterize these enzymatic transformations with atomic resolution. In tryptophan synthase, this allows us to peer along the reaction coordinates into and out of multiple intermediates. Here the protonation states complete the chemical picture for why, for example, specific inhibitors such as benzimidazole are unable to react to form a covalent bond as it is held in the wrong orientation by hydrogen bonds to βGlu109 and the charged ε-amino group of βLys87. A second goal is to extend the successes in characterizing enzymatic transformations in PLP-dependent enzymes to the β-lactamases, starting with the Toho-1 β-lactamase. Here we build on our initial chemical shift assignments and characterization of dynamics in solution to study the chemical mechanism used to inhibit antibiotics. In this application, NMR-assisted crystallography will be developed at the interface with neutron crystallography, which to date has been unable to solve the structure in the presence of an inhibitor, but where understanding the mechanism at the chemical level requires that we assign the protonation states of the key active site acid/base catalytic residues.


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 = $443,386 )
2025	2025	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	200 UNIVERSTY OFC BUILDING	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	001	4	7/25/2025	NON-COMPETING CONTINUATION	$443,386
2025	2024	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	200 UNIVERSTY OFC BUILDING	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	000	3	11/13/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $443,386

Issue Date FY: 2024 ( Subtotal = $433,121 )
2024	2024	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	200 UNIVERSTY OFC BUILDING	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	000	3	8/16/2024	NON-COMPETING CONTINUATION	$433,121
														Subtotal = $433,121

Issue Date FY: 2023 ( Subtotal = $393,796 )
2023	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	200 UNIVERSTY OFC BUILDING	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	000	2	7/25/2023	NON-COMPETING CONTINUATION	$318,799
2023	2023	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	200 UNIVERSTY OFC BUILDING	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	001	2	8/4/2023	SUPPLEMENT FOR EXPANSION	$74,997
														Subtotal = $393,796

Issue Date FY: 2022 ( Subtotal = $378,249 )
2022	2022	REGENTS OF THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE	900 UNIVERSITY AVE	RIVERSIDE	CA	92521	RIVERSIDE	USA	Biomedical Research and Research Training	000	1	7/29/2022	NEW	$378,249
														Subtotal = $378,249

Grand Total All Awards = $1,648,552

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NMR crystallography: Imaging active site chemistry and protonation states

Award Number: R35GM145369

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: 08/01/2022

PERIOD OF PERFORMANCE END DATE: 07/31/2027

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