# Structural basis underlying the autoinhibition of the formin FHOD1 and its phosphorylation-dependent activation

## TL;DR

This study reveals how FHOD1, a formin protein, is autoinhibited and activated through phosphorylation, using structural modeling and experiments.

## Contribution

The study provides the first structural model of FHOD1's autoinhibition and phosphorylation-dependent activation mechanism.

## Key findings

- An extended polybasic region stabilizes FHOD1's autoinhibitory interactions.
- Phosphorylation at the diaphanous autoregulatory domain releases autoinhibition.
- AlphaFold3 predictions were experimentally validated for FHOD1's structural model.

## Abstract

FHOD1 is a member of the formin protein family that plays a role in actin polymerization, thereby inducing stress fiber formation in vivo. FHOD1, like other members of the formin family, harbors the diaphanous autoregulatory domain at the C-terminal region, which engages in autoinhibitory interactions with the N-terminal diaphanous inhibitory domain. However, unlike other formins that are activated by the binding of Rho GTPases, autoinhibition of FHOD1 is released by phosphorylation at the diaphanous autoregulatory domain. The specific mechanisms underlying phosphorylation-dependent activation of FHOD1 remain to be elucidated, as the structure of the complex of the N- and C-terminal regions of FHOD1 remains unresolved. In this study, an in silico structural model of the autoinhibitory interaction of FHOD1 was developed using AlphaFold3. The predicted model indicated that an extended polybasic region, which is unique to the FHOD subfamily, stabilizes autoinhibitory interactions. This prediction was validated through an experimental analysis using site-directed mutagenesis. Furthermore, the extended region was implicated in the process of autoinhibition release, as expected from the findings of our previous experiments, which was successfully reinforced by the structural predictions of the phosphorylated model. These findings provide a structural basis for a unique autoinhibitory mode and the activation process of FHOD1 among formin family proteins and, at the same time, underscore the powerful utility of protein structure prediction for the refinement of our understanding of protein structures and their functional implications.