Distinguishing Allosteric Effects from Orthosteric Binding in Protein-Ligand Interactions

Summary

Application of amide hydrogen-deuterium exchange mass spectrometry to map interactions of low affinity fragment and ligands is demonstrated. This protocol describes a method for distinguishing orthosteric binding from allosteric changes accompanying high affinity ligand and low-affinity fragment binding to target protein, Hsp90, and finds important applications in fragment-based drug design.

Abstract

A fundamental challenge in deciphering protein-ligand interactions is distinguishing binding changes at orthosteric sites from the associated allosteric changes at distal sites, as structural data does not always reveal allostery. Ligands mediate both orthosteric and allosteric effects on target proteins and hence, in the context of screening low affinity fragments, it is important to describe fragment efficacy in terms of both direct binding and long-range allosteric responses. This presents a significant problem especially for low affinity ligands. Amide Hydrogen Deuterium Exchange Mass Spectrometry (HDXMS) is a robust method that can provide structural insights and information on conformational dynamics for both high affinity and transient protein-ligand interactions. Here, we describe the use of HDXMS on the ATPase domain of Hsp90, to parse orthosteric and allosteric effects mediated by two high affinity ligands and two low affinity fragment compounds. A comparison of deuterium exchange in ligand-bound-Hsp90 versus apo-Hsp90 was used to describe composite changes that combine both orthosteric effects and allosteric changes. Allostery can be discerned by correlating HDXMS results with structural information about orthosteric binding from crystallographic structures of protein-ligand interactions. Results from this approach indicated that fragments and ligands both mediate interactions at overlapping orthosteric sites but elicit distinct allosteric effects. However, orthosteric interactions of Hsp90 with fragments are inherently weaker due to faster dissociation rates (k_off). This approach finds important applications in fragment screening, ranking, and lead compound design in fragment-based drug discovery.

Introduction

Drug development necessitates a complete understanding of the interaction of natural ligands with their target proteins, and utilizes this information to find alternate inhibitors or activators. Traditional drug development pipelines involve a high throughput screening (HTS) strategy to identify lead compounds¹. An alternative strategy is to use fragments as building blocks for lead compound generation. These have multiple advantages over traditional HTS strategies, including but not limited to, being intellectual property-free, optimizable, and modular². Fragments are defined as small chemical compounds (<300 Da) wh....

Protocol

1. Preparing D ₂ O buffer, Quench and Hsp90 Protein Solutions

1. Prepare 100 µM Hsp90 protein solution (expressed in E. coli ²²) in Hsp90 aqueous buffer (20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 300 mM NaCl, 10% (v/v) glycerol, 0.5 mM TCEP (tris(2-carboxyethyl)phosphine), pH 7.5).
2. Prepare Hsp90 D₂O buffer by vacuum evaporation of the Hsp90 aqueous buffer. Dry the Hsp90 aqueous buffer by vacuum evaporation to remove H

Discussion

Critical steps in the protocol: It is essential that the pH of solutions, including protein buffers and LC-solutions, are all maintained at a pH of 2.5 to minimize loss of deuterium labelling. It is also critical that deuterium exchange experiments be carried out at saturating concentrations of ligands to maintain a homogenous population of ligand-bound protein. This can be estimated from the ligands' or fragments' dissociation constants and need to be consistent among all fragment-protein deuter.......

Materials

Name	Company	Catalog Number	Comments
Deuterium Oxide	Cambridge Isotope, Tewksbury, MA	DLM-6-1000
HEPES Buffer	Sigma Aldrich, St. Louis, MO	H3375 SIGMA
Glycerol	Sigma Aldrich, St. Louis, MO	G5516
NaCl	Sigma Aldrich, St. Louis, MO	S7653
TCEP	Sigma Aldrich, St. Louis, MO	C4706
DMSO	Sigma Aldrich, St. Louis, MO	D8418
TFA	Sigma Aldrich, St. Louis, MO	302031
FA	Fisher Scientific, Singapore	A117-50
Glu-fibrinogen	Sigma Aldrich, St. Louis, MO	F3261
Leucine-Enkaphalin	Waters, Milford, MA	186006013
ACN	Sigma Aldrich, St. Louis, MO	34851
pNIC28-Bsa4 vector	Addgene, Cambridge, MA	26103
BL21(DE3) E. coli strain	Merck-millipore Novagen, Singapore	69450
Terrific Broth medium	Sigma Aldrich, St. Louis, MO	T9179
Kanamycin	Sigma Aldrich, St. Louis, MO	60615
chloramphenicol	Sigma Aldrich, St. Louis, MO	C0378
isopropyl β-D-thiogalactopyranoside (IPTG)	Sigma Aldrich, St. Louis, MO	IPTG-RO ROCHE
imidazole	Sigma Aldrich, St. Louis, MO	I5513
Protease Inhibitor Mixture Set III, EDTA free	Merck-Millipore, Singapore	539134
Vibra-Cell processor	Sonics & Materials Inc., Newtown, CT	VC 505 / VC 750
nickel-nitrilotriacetic acid Superflow resin	Qiagen Inc., Valencia, CA	30410
HiLoad 16/60 Superdex-200 column	GE Healthcare, Waukesha, WI	28989335
Vivaspin 20 filter concentrators	GE Healthcare, Waukesha, WI	28932360
Poroszyme Immobilized Pepsin Cartridge, 2.1 mm x 30 mm	Thermofischer, Sigapore	2313100
ACQUITY UPLC BEH C18 Column	Waters, Milford, MA	186002350
ACQUITY UPLC BEH C18 VanGuard Pre-column	Waters, Milford, MA	186003975
nanoAcquity HDX sample manager	Waters, Milford, MA
Synapt G1 ESI mass spectrometer	Waters, Milford, MA
nanoAcquity Auxillary Solvent Manager	Waters, Milford, MA
nanoAcquity Binary Solvent manager	Waters, Milford, MA