A tool set to map allosteric networks through the NMR chemical shift covariance analysis.

Stephen Boulton,Rajeevan Selvaratnam,Giuseppe Melacini,Madoka Akimoto,Amir Bashiri

doi:10.1038/srep07306

Stephen Boulton, Rajeevan Selvaratnam + Show 3 more

Open Access

https://doi.org/10.1038/srep07306

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Journal: Scientific Reports	Publication Date: Dec 8, 2014
Citations: 63	License type: CC BY 4.0

Affiliation: McMaster University

Abstract

Allostery is an essential regulatory mechanism of biological function. Allosteric sites are also pharmacologically relevant as they are often targeted with higher selectivity than orthosteric sites. However, a comprehensive map of allosteric sites poses experimental challenges because allostery is driven not only by structural changes, but also by modulations in dynamics that typically remain elusive to classical structure determination methods. An avenue to overcome these challenges is provided by the NMR chemical shift covariance analysis (CHESCA), as chemical shifts are exquisitely sensitive to redistributions in dynamic conformational ensembles. Here, we propose a set of complementary CHESCA algorithms designed to reliably detect allosteric networks with minimal occurrences of false positives or negatives. The proposed CHESCA toolset was tested for two allosteric proteins (PKA and EPAC) and is expected to complement traditional comparative structural analyses in the comprehensive identification of functionally relevant allosteric sites, including those in otherwise elusive partially unstructured regions.

Highlights

Allostery is an essential regulatory mechanism of biological function
The chemical shift projection analysis (CHESPA) analysis of Rp-cAMPS was used to identify false positives in the allosteric clusters defined through CHEmical Shift Covariance Analysis (CHESCA)-SL as applied to RIa 91-244, which spans the critical cAMP binding domain (CBD) of PKA
Residues sampling primarily the allosteric inactive vs. active equilibrium are expected to exhibit ppm changes reflecting an opposite shift in the activation equilibrium relative to cAMP, whereas residues affected by Rp binding but not allosteric conformational changes would experience chemical shift changes similar to cAMP (X . 0) as well as unique ppm shifts influenced by the replacement of the equatorial phosphate oxygen with a bulkier sulphur atom in Rp (i.e. NNEs)

Summary

Introduction

Allostery is an essential regulatory mechanism of biological function. Allosteric sites are pharmacologically relevant as they are often targeted with higher selectivity than orthosteric sites. In one of the prototypical allosteric systems, i.e. the regulatory subunit of Protein Kinase A (PKA R), activation is controlled by a dynamic equilibrium between inactive and active conformations that differ at the binding site of the allosteric effector, i.e. cAMP, and at remote loci essential for inhibition of the catalytic subunit (Figure 1A)[7,8,12,13]. The linker was later found to elicit state selective interactions that allosterically couple it to cAMP12 For both EPAC and PKA R, these otherwise elusive dynamic allosteric sites were detected using an alternative approach known as the CHEmical Shift Covariance Analysis (CHESCA)[12,25]. CHESCA has been applied to other systems, revealing amino acid networks underlying enzyme catalysis and inhibition, which have been confirmed by independent mutational analyses, and shows promise for in vivo applications[12,25,29,30,31,32,33,34,35]

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