Thermal Adaptation of Cytosolic Malate Dehydrogenase Revealed by Deep Learning and Coevolutionary Analysis.

Protein evolution has shaped enzymes that maintain stability and function across diverse thermal environments. While sequence variation, thermal stability and conformational dynamics are known to influence an enzyme's thermal adaptation, how these factors collectively govern stability and function across diverse temperatures remains unresolved. Cytosolic malate dehydrogenase (cMDH), a citric acid cycle enzyme, is an ideal model for studying these mechanisms due to its temperature-sensitive flexibility and broad presence in species from diverse thermal environments. In this study, we employ techniques inspired by deep learning and statistical mechanics to uncover how sequence variation and conformational dynamics shape patterns of cMDH's thermal adaptation. By integrating coevolutionary models with variational autoencoders (VAE), we generate a latent generative landscape (LGL) of the cMDH sequence space, enabling us to explore mutational pathways and predict fitness using direct coupling analysis (DCA). Structure predictions via AlphaFold and molecular dynamics simulations further illuminate how variations in hydrophobic interactions and conformational flexibility contribute to the thermal stability of warm- and cold-adapted cMDH orthologs. Notably, we identify the ratio of hydrophobic contacts between two regions as a predictive order parameter for thermal stability features, providing a quantitative metric for understanding cMDH dynamics across temperatures. The integrative computational framework employed in this study provides mechanistic insights into protein adaptation at both sequence and structural levels, offering unique perspectives on the evolution of thermal stability and creating avenues for the rational design of proteins with optimized thermal properties.