Light-regulated allosteric switch enables temporal and subcellular control of enzyme activity.

Mark Shaaya,Forest M White,Shahzeb Khan,Jordan Fauser,Jason E Conage-Pough,Jalees Rehman,Vincent Huyot,Andrei V Karginov,Cameron T Flower,Denis Tsygankov,Pradeep Kota,Viswanathan Natarajan,Martin Brennan,Anastasia Zhurikhina,Jacob Matsche

doi:10.7554/elife.60647

Abstract

Engineered allosteric regulation of protein activity provides significant advantages for the development of robust and broadly applicable tools. However, the application of allosteric switches in optogenetics has been scarce and suffers from critical limitations. Here, we report an optogenetic approach that utilizes an engineered Light-Regulated (LightR) allosteric switch module to achieve tight spatiotemporal control of enzymatic activity. Using the tyrosine kinase Src as a model, we demonstrate efficient regulation of the kinase and identify temporally distinct signaling responses ranging from seconds to minutes. LightR-Src off-kinetics can be tuned by modulating the LightR photoconversion cycle. A fast cycling variant enables the stimulation of transient pulses and local regulation of activity in a selected region of a cell. The design of the LightR module ensures broad applicability of the tool, as we demonstrate by achieving light-mediated regulation of Abl and bRaf kinases as well as Cre recombinase.

Highlights

Dissection of biological processes is greatly aided by optogenetic methods that allow researchers to mimic and manipulate the function of individual proteins
To modulate kinase activity with light, we engineered a Light-Regulated (LightR) domain that can potentially function as an allosteric switch when inserted into a catalytic domain of a kinase
The opening of the LightR clamp could increase the distance between its N- and C- termini up to approximately 25 A, which should distort the native structure of the catalytic domain and thereby inactivate the enzyme

Summary

Introduction

Dissection of biological processes is greatly aided by optogenetic methods that allow researchers to mimic and manipulate the function of individual proteins. A few existing strategies achieve direct regulation of enzymatic activity, but they either lack the ability to activate enzymes locally within a cell, or they are difficult to apply to multiple enzyme classes Biochemistry and Chemical Biology Cell Biology eLife digest Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell

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