Domain Insertion Permissibility is a Measure of Engineerable Allostery in Ion Channels

Abstract

Allostery is a fundamental principle of protein regulation that remains poorly understood and hard to engineer, in particular in ion channels. Here we use human Inward Rectifier K+ Channel Kir2.1 to establish domain insertion permissibility as a new experimental paradigm to identify engineerable allosteric sites. We find that permissibility is best explained by dynamic protein properties, such as conformational flexibility. Many allosterically regulated sites in Kir2.1 or sites equivalent to those regulated in homologs, such as G-protein-gated inward rectifier K+ channels (GIRK), have differential permissibility; that is, for these sites permissibility depends on the structural properties of the inserted domain. Our data and the well-established link between protein dynamics and allostery led us to propose that differential permissibility is a metric of both existing and latent allostery in Kir2.1. In support of this notion, inserting light-switchable domains into either existing or latent allosteric sites, but not elsewhere, renders Kir2.1 activity sensitive to light.