Molecular function limits divergent protein evolution on planetary timescales

Sequence analyses demonstrate that even after billions of years of independent evolution, protein orthologs are still diverging from each other. Although the sequence and functional evolution of proteins have been studied in detail, it is currently unknown whether the requirement to continuously maintain the same molecular function imposes an effective limit on protein divergence. Here we investigate this fundamental question using a combined computational and experimental analysis of orthologous enzymes with the same molecular function. Interestingly, our results demonstrate that the mutual divergence rates of orthologous enzymes substantially decrease after ~1-2 billion years of independent evolution. As a result of this slowdown, which is observed well above the levels of detectible sequence homology, the sequence and structural similarities between ancient orthologs have not substantially decreased during the last billion years. We find that conservation of molecular function constrains the maximum divergence of all protein regions, including those distant from the active site residues. By experimentally characterizing the growth rates of all amino acid substitutions in the E. coli FolA protein, we delineate two stages of long-term sequence divergence with different contributions from sites with small and large fitness effects. Overall, our analysis suggests that the vast majority of ancient enzyme orthologs are unlikely to diverge, at least on planetary timescales, beyond ~25% sequence identity.