Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases

Thi Thanh Quynh Tran; Chitra Narayanan; Andrea N Loes; Timothy H Click; N T Hang Pham; Myriam Létourneau; Michael J Harms; Charles Calmettes; Pratul K Agarwal; Nicolas Doucet

doi:10.1016/j.jbc.2024.107280

Ancestral sequence reconstruction dissects structural and functional differences among eosinophil ribonucleases

J Biol Chem. 2024 Apr 6:107280. doi: 10.1016/j.jbc.2024.107280. Online ahead of print.

Affiliations

¹ Centre Armand-Frappier Santé Biotechnologie, Institut national de la recherche scientifique (INRS), Université du Québec, Laval, QC, Canada.
² Department of Chemistry, York College, City University of New York (CUNY), Jamaica, Queens, NY, USA.
³ Institute of Molecular Biology and Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA.
⁴ Chemistry and Biochemistry, University of Mary, Bismarck, ND, USA.
⁵ Centre Armand-Frappier Santé Biotechnologie, Institut national de la recherche scientifique (INRS), Université du Québec, Laval, QC, Canada; PROTEO, the Quebec Network for Research on Protein Function, Engineering, and Applications, UQAM, Montréal, QC, Canada.
⁶ Department of Physiological Sciences and High-Performance Computing Center, Oklahoma State University, Stillwater, OK, USA.
⁷ Centre Armand-Frappier Santé Biotechnologie, Institut national de la recherche scientifique (INRS), Université du Québec, Laval, QC, Canada; PROTEO, the Quebec Network for Research on Protein Function, Engineering, and Applications, UQAM, Montréal, QC, Canada. Electronic address: nicolas.doucet@inrs.ca.

PMID: 38588810
DOI: 10.1016/j.jbc.2024.107280

Abstract

Evolutionarily conserved structural folds can give rise to diverse biological functions, yet predicting atomic-scale interactions that contribute to the emergence of novel activities within such folds remains challenging. Pancreatic-type ribonucleases illustrate this complexity, sharing a core structure that has evolved to accommodate varied functions. In this study, we used ancestral sequence reconstruction to probe evolutionary and molecular determinants that distinguish biological activities within eosinophil members of the RNase 2/3 subfamily. Our investigation unveils functional, structural, and dynamical behaviors that differentiate the evolved ancestor AncRNase from its contemporary eosinophil RNase orthologs. Leveraging the potential of ancestral reconstruction for protein engineering, we used AncRNase predictions to design a minimal 4-residue variant that transforms human RNase 2 into a chimeric enzyme endowed with the antimicrobial and cytotoxic activities of RNase 3 members. This work provides unique insights into mutational and evolutionary pathways governing structure, function, and conformational states within the eosinophil RNase subfamily, offering potential for targeted modulation of RNase-associated functions.

Keywords: RNase 2; RNase 3; X-ray crystallography; ancestral RNase; ancestral sequence reconstruction; chimeragenesis; eosinophil; eosinophil cationic protein; eosinophil-derived neurotoxin; molecular dynamics; protein evolution; structural biology.