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Comparing the functional roles of nonconserved sequence positions in homologous transcription repressors: implications for sequence/function analyses.

ABSTRACT: 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.

SUBMITTER: Tungtur S

PROVIDER: S-EPMC2813367 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Comparing the functional roles of nonconserved sequence positions in homologous transcription repressors: implications for sequence/function analyses.

Tungtur Sudheer S Meinhardt Sarah S Swint-Kruse Liskin L

Journal of molecular biology 20091008 4

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 ...[more]

PMID: 19818797

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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.

Project description:The precise placement of nucleosomes has large regulatory effects on gene expression. Recent work suggests that nucleosome placement is regulated in part by the affinity of the underlying DNA sequence for the histone octamer. Nucleosome locations are also regulated by several different ATP-dependent chromatin remodeling enzymes. This raises the question of whether DNA sequence influences the activity of chromatin remodeling enzymes. DNA sequence could most simply regulate nucleosome remodeling through its effect on nucleosome stability. In such a model, unstable nucleosomes would be remodeled faster than stable nucleosomes. It is also possible that certain DNA elements could regulate remodeling by inhibiting the interaction of nucleosomes with the remodeling enzyme. A third possibility is that DNA sequence could regulate the outcome of remodeling by influencing how reaction intermediates collapse into a particular set of stable nucleosomal positions. Here we dissect the contribution from these potential mechanisms to the activities of yeast RSC and human ACF, which are representative members of two major classes of remodeling complexes. We find that varying the histone-DNA affinity over 3 orders of magnitude has negligible effects on the rates of nucleosome remodeling and ATP hydrolysis by these two enzymes. This suggests that the rate-limiting step for nucleosome remodeling may not involve the disruption of histone-DNA contacts. We further find that a specific curved DNA element previously hypothesized to inhibit ACF activity does not inhibit substrate binding or remodeling by ACF. The element, however, does influence the distribution of nucleosome positions generated by ACF. Our data support a model in which remodeling enzymes move nucleosomes to new locations by a general sequence-independent mechanism. However, consequent to the rate-limiting remodeling step, the local DNA sequence promotes a collapse of remodeling intermediates into highly resolved positions that are dictated by thermodynamic differences between adjacent positions.

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Comparing the functional roles of nonconserved sequence positions in homologous transcription repressors: implications for sequence/function analyses.

Publications

Comparing the functional roles of nonconserved sequence positions in homologous transcription repressors: implications for sequence/function analyses.

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