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Assessing the functional roles of coevolving PHD finger residues
Journal article   Peer reviewed

Assessing the functional roles of coevolving PHD finger residues

Shraddha Basu, Ujwal Subedi, Marco Tonelli, Maral Afshinpour, Nitija Tiwari, Ernesto J. Fuentes and Suvobrata Chakravarty
Protein science, Vol.33(7), e5065
07/2024
DOI: 10.1002/pro.5065
PMCID: PMC11201814
PMID: 38923615
url
https://www.ncbi.nlm.nih.gov/pmc/articles/11201814View
Open Access

Abstract

Although in silico folding based on coevolving residue constraints in the deep‐learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well‐known histone reader with distinct subtypes containing subtype‐specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model‐based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.
 AlphaFold2 coevolutionary signal coevolving residues protein family protein stability proteolysis sequence design

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