# BTK autoinhibition analyzed by high-throughput swaps of SH2 domains

## TL;DR

This study explores how Bruton’s Tyrosine Kinase (BTK) is autoinhibited without a clear latch mechanism, using high-throughput SH2 domain swaps to uncover stabilizing interactions.

## Contribution

The study reveals that distributed electrostatic interactions between SH2 and kinase domains stabilize BTK autoinhibition, unlike specialized latching mechanisms in related kinases.

## Key findings

- Many SH2 domains can substitute into BTK and increase fitness by disrupting autoinhibition.
- Electrostatic interactions between SH2 and kinase domains stabilize BTK's autoinhibited state.
- Autoinhibition in BTK relies on specific evolutionary refinements rather than generic SH2 domains.

## Abstract

Bruton’s Tyrosine Kinase (BTK) is an important target in cancer treatment, motivating studies into its mechanism. Like many other tyrosine kinases, BTK contains an SH2 domain that binds to phosphotyrosine residues. However, unlike kinases in the closely related Src and Abl families, BTK lacks an obvious intramolecular latch that stabilizes its autoinhibited state. Using high-throughput methods that enable measurement of the effects of hundreds of SH2-domain replacements, we find that the SH2 domain is crucial for stabilizing the autoinhibited state of BTK. Electrostatic interactions between the SH2 and kinase domains serve to stabilize the SH2 domain in an inhibitory conformation, suggesting that specialized latching mechanisms were a later evolutionary refinement.

Bruton’s Tyrosine Kinase (BTK), a Tec-family tyrosine kinase, resembles the Src and Abl kinases in that an SH2-SH3 module regulates the activity of the kinase domain, principally through an inhibitory interaction between the SH3 and kinase domains. In Src-family kinases, phosphorylation of a C-terminal tail latches the SH2 domain onto the kinase domain, stabilizing the inhibitory conformation of the SH3 domain; in Abl, interaction between the kinase domain and a myristoyl group on the N-terminal segment provides a similar latching function. The structure of autoinhibited BTK resembles that of the Src and Abl kinases, but BTK lacks an obvious SH2-kinase latch. To assess the role of the SH2 domain in autoinhibition of BTK, we generated hundreds of chimeric BTK molecules in which the native SH2 domain is replaced by other SH2 domains. We measured the fitness of these chimeric proteins using a high-throughput assay in T and B cells. Surprisingly, many SH2 domains increased fitness when substituted into BTK. Analysis of one set of chimeric proteins indicates that the increase in fitness stems from the ability of the substituted SH2 domains to disrupt BTK autoinhibition while maintaining phosphotyrosine targeting. Thus, although BTK lacks a specialized latch, distributed interactions between the SH2 and kinase domains stabilize the autoinhibitory conformation of BTK. While phosphotyrosine recognition can be conferred on BTK by evolutionarily distant SH2 domains, autoinhibition requires specific interactions with the kinase domain that arose through evolutionary refinement of the regulatory mechanism, and is less easily mimicked by heterologous SH2 domains.