An expanded allosteric network in PTP1B by multitemperature crystallography, fragment screening, and covalent tethering.

Daniel A Keedy,José Brandão-Neto,Nicholas M Pearce,Frank Von Delft,Zachary B Hill,Justin T Biel,T Justin Rettenmaier,James A Wells,James S Fraser,Emily Kang

doi:10.7554/elife.36307

Abstract

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

Highlights

Proteins are collections of atoms that are mechanically coupled to one another, which gives rise to coordinated motions within the constraints of the folded structure
To prioritize putative allosteric sites rather than benign binding sites, we focused on the subset of fragment-binding sites that were conformationally coupled to the active site based on multitemperature crystallography of apo protein tyrosine phosphatase 1B (PTP1B)
Identifying allosterically coupled residues with multitemperature crystallography To identify allosteric sites in PTP1B that can communicate with the active site, we searched for regions of the protein whose conformational heterogeneity is coupled to that of the active site

Summary

Introduction

Proteins are collections of atoms that are mechanically coupled to one another, which gives rise to coordinated motions within the constraints of the folded structure. These motions are critical for many processes in molecular biology, including small-molecule and protein:protein binding interactions, catalytic cycles in enzymes, and allosteric communication between active sites and distal regulatory sites. Allostery in particular is recognized to occur in classical oligomeric proteins like hemoglobin and in monomers – and may be inherent to most protein structures (Motlagh et al, 2014; Gunasekaran et al, 2004). We do not yet understand at a fundamental level how mechanically coupled atoms underlie communication through protein structures, which prevents us from mapping their intrinsic allosteric ‘circuitry’.

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Conclusion