Collective Dynamics Differentiates Functional Divergence in Protein Evolution

Abstract

Protein evolution is most commonly studied by analyzing related protein sequences and generating ancestral sequences through Bayesian and Maximum Likelihood methods, and/or by resurrecting ancestral proteins in the lab and performing ligand binding studies to determine function. Structural and dynamic evolution have largely been left out of molecular evolution studies. Here we incorporate both structure and dynamics to elucidate the molecular principles behind the divergence in the evolutionary path of the steroid receptor proteins. We determine the likely structure of three evolutionarily diverged ancestral steroid receptor proteins using the Zipping and Assembly Method with FRODA (ZAMF). Our predictions are within ∼2.7 Å all-atom RMSD of the respective crystal structures of the ancestral steroid receptors. Beyond static structure prediction, a particular feature of ZAMF is that it generates protein dynamics information. We investigate the differences in conformational dynamics of diverged proteins by obtaining the most collective motion through essential dynamics. Strikingly, our analysis shows that evolutionarily diverged proteins of the same family do not share the same dynamic subspace, while those sharing the same function are simultaneously clustered together and distant from those, that have functionally diverged. Dynamic analysis also enables those mutations that most affect dynamics to be identified. It correctly predicts all mutations (functional and permissive) necessary to evolve new function and ∼60% of permissive mutations necessary to recover ancestral function.

Author Summary

Proteins are remarkable machines of the living systems that show diverse biochemical functions. Biochemical diversity has grown over time via molecular evolution. In order to understand how diversity arose, it is fundamental to understand how the earliest proteins evolved and served as templates for the present diverse proteome. The one sequence - one structure - one function paradigm is being extended to a new view: an ensemble of different conformations in equilibrium can evolve new function and the analysis of inherent structural dynamics is crucial to give a more complete understanding of protein evolution. Therefore, we aim to bring structural dynamics into protein evolution through our zipping and assembly method with FRODA. (ZAMF). We apply ZAMF to simultaneously obtain structures and structural dynamics of three ancestral sequences of steroid receptor proteins. By comparative dynamics analysis among the three ancestral steroid hormone receptors: (i) we show that changes in the structural dynamics indicates functional divergence and (ii) we identify all functionally critical and most of the permissive mutations necessary to evolve new function. Overall, all these findings suggest that conformational dynamics may play an important role where new functions evolve through novel molecular interactions.