Rheostat functional outcomes occur when substitutions are introduced at nonconserved positions that diverge with speciation

When amino acids vary during evolution, the outcome can be functionally neutral or biologically-important. We previously found that substituting a subset of nonconserved positions, rheostat positions, can have surprising effects on protein function. Since changes at rheostat positions can facilitate functional evolution or cause disease, more examples are needed to understand their unique biophysical characteristics. Here, we explored whether phylogenetic patterns of change in multiple sequence alignments (such as positions with subfamily specific conservation) predict the locations of functional rheostat positions. To that end, we experimentally tested eight phylogenetic positions in human liver pyruvate kinase (hLPYK), using 10-15 substitutions per position and biochemical assays that yielded five functional parameters. Five positions were strongly rheostatic and three were non-neutral. To test the corollary that positions with low phylogenetic scores were not rheostat positions, we combined these phylogenetic positions with previously-identified hLPYK rheostat, toggle (most substitution abolished function), and neutral (all substitutions were like wild-type) positions. Despite representing 428 variants, this set of 33 positions was poorly statistically powered. Thus, we turned to the in vivo phenotypic dataset for E. coli lactose repressor protein (LacI), which comprised 12-13 substitutions at 329 positions and could be used to identify rheostat, toggle, and neutral positions. Combined hLPYK and LacI results show that positions with strong phylogenetic patterns of change are more likely to exhibit rheostat substitution outcomes than neutral or toggle outcomes. Furthermore, phylogenetic patterns were more successful at identifying rheostat positions than were co-evolutionary or eigenvector centrality measures of evolutionary change.

Automated summary

Variations in amino acids during evolution can have functionally neutral or biologically important outcomes, with changes at rheostat positions having significant effects on protein function. Experimental testing revealed that positions with strong phylogenetic patterns are more likely to exhibit rheostat substitution outcomes, rather than neutral or toggle outcomes.