# ALLOSTERIC IMPACT OF NON-ACTIVE-SITE MUTATIONS ON ENZYMATIC FUNCTION

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2020 · $299,281

## Abstract

Antibiotic-resistant infections kill tens of thousands of Americans and cost our nation billions of dollars every
year. β-lactamase enzymes are one of the most common sources of resistance and are capable of quickly
evolving the ability to degrade new β-lactam antibiotics as they are introduced. Surprisingly, many of the
mutations that confer β-lactamases with new functions are far from the enzyme's active site and have little
effect on the structure of the active site, as observed by x-ray crystallography. Such non-active site (NAS)
mutations also appear frequently in other contexts, such as the evolution of other forms of drug resistance and
directed evolution studies. Understanding how NAS mutations allosterically impact distant sites would provide
a basis for predicting new forms of drug resistance and designing allosteric drugs to combat diseases like
antibiotic-resistant infections. The objective of this proposal is to understand how NAS mutations confer β-
lactamases with activity against new substrates. A predictive understanding of NAS mutations remains elusive
because of the ruggedness of proteins' energy landscapes and the great diversity of mechanisms that couple
distant residues, including both concerted structural changes and correlations between the dynamics of
different residues. These obstacles will be overcome by integrating novel computational methods with in vitro
and in vivo experiments to converge on a quantitative understanding of the full spectrum of correlated
fluctuations responsible for allosteric coupling. For example, the research team will apply new methods they
developed to facilitate comprehensive sampling of proteins' energy landscapes, such as their FAST algorithm
for leveraging Markov State Models (MSMs) to efficiently sample conformations with pre-specified features. In
Aim 1, these methods will be used to identify what features of β-lactamase's structure and dynamics give rise
to new activities by comparing models for variants with different activities against the antibiotic cefotaxime. In
aim 2, new methods for identifying both concerted structural changes and correlations between the dynamics
of different residues will be developed. These methods will be used to predict new sites where NAS mutations
can alter activities of β-lactamases. To test insights from each aim, mutations will be designed to confer β-
lactamases with new activities. Then experiments will be performed to test 1) whether these mutations have
the intended impact on the activities of β-lactamases and 2) whether the designed variants are capable of
protecting bacteria from the target antibiotic. Completion of this work will result in a general framework for
understanding allosteric communication that will serve as a basis for future efforts to predict drug resistance,
design new antibiotics that allosterically inhibit their targets, and manipulate allostery in other systems.

## Key facts

- **NIH application ID:** 9977221
- **Project number:** 5R01GM124007-04
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Gregory Bowman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $299,281
- **Award type:** 5
- **Project period:** 2017-09-05 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9977221

## Citation

> US National Institutes of Health, RePORTER application 9977221, ALLOSTERIC IMPACT OF NON-ACTIVE-SITE MUTATIONS ON ENZYMATIC FUNCTION (5R01GM124007-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9977221. Licensed CC0.

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