PYK-SubstitutionOME: an integrated database containing allosteric coupling, ligand affinity and mutational, structural, pathological, bioinformatic and computational information about pyruvate kinase isozymes.

Interpreting changes in patient genomes, understanding how viruses evolve and engineering novel protein function all depend on accurately predicting the functional outcomes that arise from amino acid substitutions. To that end, the development of first-generation prediction algorithms was guided by historic experimental datasets. However, these datasets were heavily biased toward substitutions at positions that have not changed much throughout evolution (i.e. conserved). Although newer datasets include substitutions at positions that span a range of evolutionary conservation scores, these data are largely derived from assays that agglomerate multiple aspects of function. To facilitate predictions from the foundational chemical properties of proteins, large substitution databases with biochemical characterizations of function are needed. We report here a database derived from mutational, biochemical, bioinformatic, structural, pathological and computational studies of a highly studied protein family-pyruvate kinase (PYK). A centerpiece of this database is the biochemical characterization-including quantitative evaluation of allosteric regulation-of the changes that accompany substitutions at positions that sample the full conservation range observed in the PYK family. We have used these data to facilitate critical advances in the foundational studies of allosteric regulation and protein evolution and as rigorous benchmarks for testing protein predictions. We trust that the collected dataset will be useful for the broader scientific community in the further development of prediction algorithms. Database URL https://github.com/djparente/PYK-DB.

Odd one out? Functional tuning of Zymomonas mobilis pyruvate kinase is narrower than its allosteric, human counterpart.

Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation ("phylogenetic" positions) were enriched for "rheostat" positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes). However, the correlation was moderate, which could result from multiple biophysical constraints acting on the same position during evolution and/or various sources of noise. To further examine this correlation, we here tested Zymomonas mobilis PYK (ZmPYK), which has <65% sequence identity to any other PYK sequence. Twenty-six ZmPYK positions were selected based on their phylogenetic scores, substituted with multiple amino acids, and assessed for changes in Kapp-PEP . Although we expected to identify multiple, strong rheostat positions, only one moderate rheostat position was detected. Instead, nearly half of the 271 ZmPYK variants were inactive and most others showed near wild-type function. Indeed, for the active ZmPYK variants, the total range of Kapp,PEP values ("tunability") was 40-fold less than that observed for hLPYK variants. The combined functional studies and sequence comparisons suggest that ZmPYK has evolved functional and/or structural attributes that differ from the rest of the family. We hypothesize that including such "orphan" sequences in MSA analyses obscures the correlations used to predict rheostat positions. Finally, results raise the intriguing biophysical question as to how the same protein fold can support rheostat positions in one homolog but not another.

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.

Rheostat positions: A new classification of protein positions relevant to pharmacogenomics.

To achieve the full potential of pharmacogenomics, one must accurately predict the functional out comes that arise from amino acid substitutions in proteins. Classically, researchers have focused on understanding the consequences of individual substitutions. However, literature surveys have shown that most substitutions were created at evolutionarily conserved positions. Awareness of this bias leads to a shift in perspective, from considering the outcomes of individual substitutions to understanding the roles of individual protein positions. Conserved positions tend to act as "toggle" switches, with most substitutions abolishing function. However, nonconserved positions have been found equally capable of affecting protein function. Indeed, many nonconserved positions act like functional dimmer switches ("rheostat" positions): This is revealed when multiple substitutions are made at a single position. Each substitution has a different functional outcome; the set of substitutions spans arange of outcomes. Finally, some nonconserved positions appear neutral, capable of accommodating all amino acid types without modifying function. This manuscript reviews the currently-known properties of rheost at positions, with examples shown for pyruvate kinase, organic anion transporting polypeptide 1B1, the beta-lactamase inhibitory protein, and angiotensin-converting enzyme 2. Outcomes observed for rheostat positions have implications for the rational design of drug analogs and allosteric drugs. Furthermore, this new framework - comprising three types of protein positions - provides a new approach to interpreting disease and population-based databases of amino acid changes. In conclusion, although a full understanding of substitution out comes at rheostat positions poses a challenge, utilization of this new frame of reference will further advance the application of pharmacogenomics.