# Allosteric Regulation of Actin Capping Protein: Mechanism and Significance

> **NIH NIH R35** · WASHINGTON UNIVERSITY · 2022 · $552,436

## Abstract

Project Summary/Abstract
 Actin assembly underlies and drives many biological phenomena. Barbed ends
of actin filaments, the major sites of polymerization, are controlled by the heterodimeric
actin capping protein (CP). CP is regulated by the direct binding of CPI-motif proteins
and the protein V-1. These two classes of regulators, CPI-motif proteins and V-1, bind
to opposite sides of CP, and they induce conformational changes in CP that
allosterically antagonize the binding of the other class. We are studying the molecular
biophysical mechanism of these allosteric regulators. We are also studying the
physiological function of CP and its regulators, using biochemical reconstitution with
purified components, along with molecular genetic perturbations of living cells.
 Our biochemical studies will test a novel hypothesis for how CP regulators
function in cells. Cells contain stoichiometric amounts of V-1 in micromolar
concentrations, sufficient to inhibit nearly all of the cellular CP. V-1 is highly diffusible,
and V-1 sterically blocks the ability of CP to cap actin filaments. CPI-motif proteins are
targeted to membranes, and their CPI motifs allosterically induce the dissociation of V-
1, thus activating CP locally at the membrane.
 We are testing this hypothesis by determining the molecular biophysical
mechanism of the allostery, and by testing the functions of the CPI-motif proteins with
respect to cell motility and migration. We have discovered key differences in the
biochemical activities of different families of CPI-motif proteins, and we are now using
that information to investigate the allosteric mechanism, by combining single-molecule
FRET measurements with molecular dynamics simulations. In addition, we are using
purified proteins and lipids in a biochemical reconstitution system that induces actin
assembly at a surface, thereby mimicking actin polymerization at cellular membranes.
We are also using our discoveries about biochemical activities of CPI motifs to test
cellular functions of CPI-motif proteins, using chimeras constructed from different CPI-
motif proteins with molecular genetic perturbations and real-time movies of the motility
phenomena of living cells. Our cell motility assays employ a system of endothelial cell
monolayers with transmigrating immune and cancer cells, mimicking the physiological
process of transendothelial migration.

## Key facts

- **NIH application ID:** 10330809
- **Project number:** 1R35GM144082-01
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** JOHN A COOPER
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $552,436
- **Award type:** 1
- **Project period:** 2022-01-19 → 2026-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10330809

## Citation

> US National Institutes of Health, RePORTER application 10330809, Allosteric Regulation of Actin Capping Protein: Mechanism and Significance (1R35GM144082-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10330809. Licensed CC0.

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