Positively Selected Sites in Cetacean Myoglobins Contribute to Protein Stability

Abstract

Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10–20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity of Mbs is not significantly different among mammals (with typical oxygenation constants of ∼0.8–1.2 µM−1), folding stabilities of cetacean Mbs are ∼2–4 kcal/mol higher than for terrestrial Mbs. Using ancestral sequence reconstruction, maximum likelihood and Bayesian tests to describe the evolution of cetacean Mbs, and experimentally calibrated computation of stability effects of mutations, we observe accelerated evolution in cetaceans and identify seven positively selected sites in Mb. Overall, these sites contribute to Mb stabilization with a conditional probability of 0.8. We observe a correlation between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected sites that occur later act against other destabilizing mutations to maintain stability across the clade, except for the shallow divers, where late stability relaxation occurs, probably due to the shorter aerobic dive limits of these species. The three main positively selected sites 66, 5, and 35 undergo changes that favor hydrophobic folding, structural integrity, and intra-helical hydrogen bonds.

Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes

“…The stability of primate Mb fluctuated but did not increase systematically for apes and primates. In contrast, cetaceans Mb stability did increase by 23 kcal/mol due to the combination of many substitutions [43]; a finding confirmed by experimental measures of whale vs. human Mb stability [86]. This provides a comparative control where stability of another protein, Mb, is enhanced in cetaceans rather than apes, but stability of SOD1 it is enhanced in great apes.…”

“…There are now SOD1 sequences available that represent all key families, including Hominidae (great apes), Hylobatidae (lesser apes), old world monkeys, and several families of the new world monkeys, as well as sequences from the other suborder of primates Strepsirrhini (galago) and from the closest related order of Scandentia, represented by the tree shrew; this makes the phylogeny well sampled. Such "protein-explicit phylogeny" was previously applied to myoglobin of diving cetaceans [43] and involves mapping specific protein orthologs onto established species trees to deduce the ancestors of the evolved protein, enforced by the species phylogeny. Subsequently, the ancient proteins are studied to identify protein properties that have changed during evolution [43] [44].…”

“…Such "protein-explicit phylogeny" was previously applied to myoglobin of diving cetaceans [43] and involves mapping specific protein orthologs onto established species trees to deduce the ancestors of the evolved protein, enforced by the species phylogeny. Subsequently, the ancient proteins are studied to identify protein properties that have changed during evolution [43] [44]. This approach reveals insight into the molecular causes and types of recent selection pressure on genes.…”

“…Specifically, myoglobin (Mb), a widely studied and highly expressed mammalian protein, has been studied by similar approaches [43], also using the consensus mammalian tree by Meredith et al [39]. The stability of primate Mb fluctuated but did not increase systematically for apes and primates.…”

“…As the template human SOD1 has "old" residues that are sometimes "new" in the phylogeny, changes were assumed to be reversible, as they ideally should be; this approach has previously been shown to be accurate for I-Mutant and Popmusic [43][44], but not generally [68]. In cases where neither the new or old residue was present in the template 2C9V structure but rather a third residue X, we used the fact that free energy is a state function and computed the changes as ∆∆G (new from X) -∆∆G (old from X).…”