# Studies of Allostery between Multi-domain Proteins and Nucleic Acid Complexes

> **NIH NIH R01** · YALE UNIVERSITY · 2021 · $381,800

## Abstract

Project Summary
 The PI Batista from Yale and co-investigators (Lisi, Brown University, and Palermo, UC Riverside) will
investigate allosteric pathways in the CRISPR-Cas9 system – composed of the multi-domain endonuclease
Cas9 in complex with RNA and DNA. The system allows for studies of long-range signaling critical for allosteric
mechanisms that achieve enhanced selectively and tunability of the protein/nucleic acid complex response.
CRISPR-Cas9 is an innovative therapeutic tool with widely demonstrated capabilities for genome editing. An
outstanding challenge of great research interest is to develop a detailed understanding of allosteric signals in
CRISPR-Cas9 responsible for the DNA editing capability. Such understanding would have profound implications
for bioengineering and precision medicine, as well as for establishing modern paradigms of allosteric regulation
in protein/nucleic acid machines. A substantial hurdle in investigating the mechanisms of large protein/nucleic
acid complexes is the inherent difficulty of adapting experimental and computational methodologies to capture
the intrinsic flexibility of these structures essential for functionality. We propose to implement a synergistic
approach of solution NMR and molecular dynamics (MD) in combination with established and novel methods for
analysis of allosteric networks to elucidate the structural and dynamic determinants of allosteric signaling in
CRISPR-Cas9. We have recently identified a pathway of dynamic communication connecting multiple domains
of Cas9 through millisecond timescale motion that spans its critical nucleases, consistent with a regulatory signal
proposed through experimental characterization. Thus, the following hypotheses guide our specific aims: (i) A
well-defined allosteric pathway controls the CRISPR-Cas9 functionality; (ii) The allosteric interplay between
spatially distant protein domains activates the DNA nuclease function; (iii) Modulation of the allosteric motions
through the mutation of critical residues achieves altered specificity; and (iv) Dynamically-driven signaling is an
intrinsic property of protein-nucleic acid macromolecular complexes. Our specific aims are: Aim 1: Characterize
the allosteric control of the HNH nuclease; Aim 2: Determine the allosteric pathway from HNH to RuvC and the
allosteric role of the PAM recognition sequence; and Aim 3: Characterize the effect of mutations on the allosteric
pathway. The research program involves multiple cycles of an iterative approach where, in each cycle, allosteric
pathways are explored through the analysis of differential motions probed by liquid-NMR relaxation methods and
computation (MD and network analysis), obtaining valuable information on key amino acid residues and specific
interactions responsible for transmitting structural or dynamical changes spanning the allosteric and active sites.
The resulting insight provides guidelines for the next round of studies of mutants and modulators in a joint
...

## Key facts

- **NIH application ID:** 10120147
- **Project number:** 1R01GM136815-01A1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Victor S Batista
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $381,800
- **Award type:** 1
- **Project period:** 2021-02-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10120147

## Citation

> US National Institutes of Health, RePORTER application 10120147, Studies of Allostery between Multi-domain Proteins and Nucleic Acid Complexes (1R01GM136815-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10120147. Licensed CC0.

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