Engineering Order and Cooperativity in a Disordered Protein.

Sneha Munshi,Samyuktha Ramesh,Luis A Campos,Divakar Kalivarathan,Athi N Naganathan,Hemashree Golla,Ashok Sekhar,Madhurima Kulkarni,Sandhyaa Subramanian

doi:10.1021/acs.biochem.9b00182

Abstract

Structural disorder in proteins arises from a complex interplay between weak hydrophobicity and unfavorable electrostatic interactions. The extent to which the hydrophobic effect contributes to the unique and compact native state of proteins is, however, confounded by large compensation between multiple entropic and energetic terms. Here we show that protein structural order and cooperativity arise as emergent properties upon hydrophobic substitutions in a disordered system with non-intuitive effects on folding and function. Aided by sequence-structure analysis, equilibrium, and kinetic spectroscopic studies, we engineer two hydrophobic mutations in the disordered DNA-binding domain of CytR that act synergistically, but not in isolation, to promote structure, compactness, and stability. The double mutant, with properties of a fully ordered domain, exhibits weak cooperativity with a complex and rugged conformational landscape. The mutant, however, binds cognate DNA with an affinity only marginally higher than that of the wild type, though nontrivial differences are observed in the binding to noncognate DNA. Our work provides direct experimental evidence of the dominant role of non-additive hydrophobic effects in shaping the molecular evolution of order in disordered proteins and vice versa, which could be generalized to even folded proteins with implications for protein design and functional manipulation.

Highlights

Disordered proteins (IDPs), many of which act as hubs in protein−protein interaction networks, play a vital role in cellular signaling, transcription, and regulation in eukaryotes, bacteria, and viruses
Their abundance is evidence of an evolutionary selection for disorder, the advantages of which can range from efficient regulation, high flexibility enabling simultaneous binding to multiple partners, switching functions upon post-translational modifications, and intrinsic sensitivity to solvent conditions to a large capture radius.[1−5] Many Intrinsically disordered proteins (IDPs) fold upon binding to their partner proteins and acquire a structure that is determined by both its intrinsic sequence properties and the details of the molecular surface presented by the partner
Can an IDP be engineered to fold to a compact structure via minimal hydrophobic substitutions? What is the consequence of structural ordering for the conformational landscape and the binding affinity for its partner ligand? In particular, the role of protein sequence hydrophobicity and its nonadditive effect on protein stability has been predicted and tested via multiple approaches in well-folded domains.[23−28] The non-additivity has its origins in complex solvation effects and the precise and directional nature of packing interactions that are almost always interlinked when studying folded proteins.[29−37] Disordered domains, on the other hand, can serve as excellent model systems for understanding how higher-order packing effects emerge upon mutations in the sequence, as packing interactions are minimal or non-existent to start with

Summary

Introduction

Disordered proteins (IDPs), many of which act as hubs in protein−protein interaction networks, play a vital role in cellular signaling, transcription, and regulation in eukaryotes, bacteria, and viruses. If the design of a folded variant is successful, the resulting changes in ensemble dimensions and stability will be more dramatic, providing a cleaner signal These two terms should provide a direct measure of the extent to which solvation (and the “hydrophobic effect”) contributes to polymer compaction to a folded structure (and not to a nonspecific globule) while establishing multiple weak nonlocal interactions that determine cooperativity.[38−43]. We answer these questions in this work by employing the disordered DNA-binding domain (DBD) of CytR as a model system (termed CytR hereafter),[44] a member of the LacI DBD family. We provide direct experimental evidence of the non-additive nature of hydrophobicity and experimentally demonstrate the emergent nature of order and cooperativity expected of polymeric systems

Methods

Results

Conclusion