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

Abstract

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 K app-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 K app,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.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

“…Fortunately, we empirically determined that ∼10 substitutions (semiSM) are sufficient to categorize each position as rheostat or toggle and can provide a reasonable estimate of neutrality. 2; 7; 9; 37; 57; 59 The current, semiSM CIITA experiments used 10-14 variants (including WT) for each of the seven studied positions (Table 1).…”

“…In other proteins, the full tuneable range can be accessed by a variety of single substitutions (e.g., 1 ). Furthermore, in some globular proteins, 50-100% of the tuneable functional range can be sampled by a set of substitutions at an individual "rheostat" position (e.g., [2][3][4][5][6][7][8][9] ). It is still unknown whether the existence of rheostat positions requires a classically-folded protein structure or whether they can also be present in intrinsically disordered regions (IDRs).…”

“…In other proteins, the full tuneable range can be accessed by a variety of single substitutions (e.g., 1 ). Furthermore, site-saturating and semi-saturating mutagenesis (SSM and semiSM, respectively) of single positions revealed that, in some globular proteins, substituting individual "rheostat" positions can sample 50-100% of the tuneable functional range (e.g., [2][3][4][5][6][7][8][9] ). It is still unknown whether a classically folded protein structure is a pre-requisite for the presence of rheostat positions or whether they can also be present in intrinsically disordered regions (IDRs).…”

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

“…For the seven tested positions, no correlation was observed between ConSurf scores and substitution outcomes. However, CIITA is only present in vertebrates; the other studies correlating substitution outcomes with evolutionary scores were for pyruvate kinase, which are present in all domains of life (Page et al, 2022) and LacI/ GalR homologs, which are present in almost all bacteria (Meinhardt et al, 2012(Meinhardt et al, , 2013. Both of these families are much older than CIITA, providing more opportunities to explore sequence space.…”

“…In other proteins, the full tuneable range can be accessed by a variety of single substitutions (e.g., Tang & Fenton, 2017). Furthermore, in some globular proteins, 50%–100% of the tuneable, functional range can be sampled by a set of substitutions at an individual “rheostat” position (e.g., Bantis et al, 2020; Campitelli et al, 2020, 2023; Fenton et al, 2020; Page et al, 2022; Ruggiero et al, 2021; Swint‐Kruse et al, 2021; Wu et al, 2019). It is still unknown whether the existence of rheostat positions requires a classically‐folded protein structure or whether they can also be present in intrinsically disordered regions (IDRs).…”