Engineered synthetic antibodies as probes to quantify the energetic contributions of ligand binding to conformational changes in proteins

Somnath Mukherjee,Dionne H Griffin,James R Horn,Shahir S Rizk,Malgorzata Nocula-Lugowska,Magnus Malmqvist,Sangwoo S Kim,Anthony A Kossiakoff

doi:10.1074/jbc.ra117.000656

Abstract

Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. However, it is often challenging to link ligand-induced conformational changes to a resulting biological function because it is difficult to distinguish between the energetic components associated with ligand binding and those due to structural rearrangements. Here, we used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Using maltose-binding protein (MBP) as a model system, customized phage-display selections were performed to generate sABs that stabilize MBP in different conformational states, modulating ligand-binding affinity in competitive, allosteric, or peristeric manners. We determined that the binding of a closed conformation-specific sAB (sAB-11M) to MBP in the absence of maltose is entropically driven, providing new insight into designing antibody-stabilized protein interactions. Crystal structures of sABs bound to MBP, together with biophysical data, delineate the basis of free energy differences between different conformational states and confirm the use of the sABs as energy probes for dissecting enthalpic and entropic contributions to conformational transitions. Our work provides a foundation for investigating the energetic contributions of distinct conformational dynamics to specific biological outputs. We anticipate that our approach also may be valuable for analyzing the energy landscapes of regulatory proteins controlling biological responses to environmental changes.

Highlights

Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems
Conformational changes in response to ligand binding are ubiquitous among biological processes
In this regard, exploiting the power of phage display, we have developed a versatile set of affinity reagents that can be used to investigate the energetics of these complex biological phenomena by exploiting the ability of these reagents to trap functionally important conformations

Summary

Introduction

Conformational changes in proteins due to ligand binding are ubiquitous in biological processes and are integral to many biological systems. We used a unique approach exploiting conformation-specific and regio-specific synthetic antibodies (sABs) to probe the energetic contributions of ligand binding to conformation changes. Assigning and quantifying the energetic forces that link specific conformational changes to a resulting function are only understood at a basic level This is because it is difficult to distinguish between the energetic contributions derived from the ligand-binding event and energies originating from the molecular rearrangements encoded in the conformational changes. To better understand these phenomena, we endeavored to develop a unique approach that would provide a more direct readout about how the thermodynamic relationships between ligand binding and conformational partitioning are coupled This approach involves using conformationally selective synthetic antibodies (sABs) generated by phage display as energetic probes to dissect the thermodynamic contributions of ligand-binding versus conformation changes.

Methods

Results

Conclusion