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

ABSTRACT: 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.

SUBMITTER: Swint-Kruse L

PROVIDER: S-EPMC8376419 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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

Swint-Kruse Liskin L Martin Tyler A TA Page Braelyn M BM Wu Tiffany T Gerhart Paige M PM Dougherty Larissa L LL Tang Qingling Q Parente Daniel J DJ Mosier Brian R BR Bantis Leonidas E LE Fenton Aron W AW

Protein science : a publication of the Protein Society 20210611 9

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 seque ...[more]

PMID: 34076313

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Project description:The explosion of protein sequences deduced from genetic code has led to both a problem and a potential resource: Efficient data use requires interpreting the functional impact of sequence change without experimentally characterizing each protein variant. Several groups have hypothesized that interpretation could be aided by analyzing the sequences of naturally occurring homologues. To that end, myriad sequence/function analyses have been developed to predict which conserved, semi-conserved, and nonconserved positions are functionally important. These positions must be discriminated from the nonconserved positions that are functionally silent. However, the assumptions that underlie sequence analyses are based on experimental results that are sparse and usually designed to address different questions. Here, we use three homologues from a test family common to bioinformatics-the LacI/GalR transcription repressors-to test a common assumption: If a position is functionally important for one family member, it has similar importance in all homologues. We generated experimental sequence/function information for each nonconserved position in the 18 amino acids that link the DNA-binding and regulatory domains of three LacI/GalR homologues. We find that the functional importance of each position is preserved among the three linkers, albeit to different degrees. We also find that every linker position contributes to function, which has twofold implications. (1) Since the linker positions range from highly conserved to semi-conserved to nonconserved and contribute to affinity, selectivity, and allosteric response, we assert that sequence/function analyses must identify positions in the LacI/GalR linkers to be qualified as "successful". Many analyses overlook this region since most of the residues do not directly contact ligand. (2) No position in the LacI/GalR linker is functionally silent. This finding is inconsistent with another underlying principle of many analyses: Using sequence sets to discriminate important from non-contributing positions obligates silent positions, which denotes that most homologues tolerate a variety of amino acid substitutions at the position without functional change. Instead, additional combinatorial mutants in the LacI/GalR linkers show that particular substitutions can be silent in a context-dependent manner. Thus, specific permutations of sequence change (rather than change at silent positions) would facilitate neutral drift during evolution. Finally, the combinatorial mutants also reveal functional synergy between semi- and nonconserved positions. Such functional relationships would be missed by analyses that rely primarily upon co-evolution.

Project description:The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

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

Publications

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

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