Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design.

Arun Chandramohan,Andreas Larsson,Srinath Krishnamurthy,Paer Nordlund,Ganesh S Anand,Anna Jansson,Jin Liu

doi:10.1371/journal.pcbi.1004840

Abstract

A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD).

Highlights

Ligands mediate specific interactions with proteins and alter their conformational dynamics thereby modulating their function, making them important regulators of biological processes [1]
We describe the application of amide hydrogen/deuterium exchange mass
Distinguishing Allostery from Binding in Protein-Ligand Interactions spectrometry (HDXMS) to differentiate between changes occurring at the binding site and at distal allosteric sites by combining hydrogen deuterium Exchange mass spectrometry (HDXMS) with X-ray crystallography

Summary

Introduction

Ligands mediate specific interactions with proteins and alter their conformational dynamics thereby modulating their function, making them important regulators of biological processes [1]. X-ray crystallography has been a powerful method of choice for obtaining high resolution structures of ligand-protein complexes and provide atomic level insights of ligand interactions and their binding sites. These only represent snapshot average endstates of proteins that do not always provide a complete overview of long range conformational changes. These represent observed changes that are distal from the proximal ligand binding sites as defined by high resolution structures. A major question that remains is how proximal binding effects at orthosteric sites can be distinguished from long-range conformational changes at distal sites across the protein

Methods

Results

Discussion

Conclusion