Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space

Abstract

Many genomes contain families of paralogs-proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response elementbinding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.evolution | glucocorticoid | epistasis | steroid receptors

Glucocorticoid receptor control of transcription: precision and plasticity via allostery

“…This contrasts strongly with the cooperative binding seen at canonical GBSs 25 . Furthermore, nuclear magnetic resonance (NMR) studies indicate that the D-loop residues display significant changes in chemical environment consistent with dimerization when bound to a canonical GBS, whereas they are unaffected on binding to an IR-GBS 26 . As with canonical GBSs, it has not been examined whether LBD-LBD interactions participate in GR binding to IR-GBSs.…”

“…Taken together, we infer from these in vitro studies that monomeric GR most likely binds these elements in vivo . Other 3-keto steroid receptors, MR, PR and AR, can all bind to a canonical GBS, but only GR is able to bind an IR-GBS 26 . Even the MR DBD, which shares 90% sequence identity with the GR DBD, cannot bind or repress transcription from an IR-GBS, owing to epistatic mutations that occurred during the evolution of the steroid receptor family that limit this function in all steroid receptors except GR 26 .…”

“…Additionally, NMR analyses revealed allosteric communications between the dimerized GR monomers at canonical GBSs 19 , and molecular dynamics simulations (for further details of the method, see Supplementary information S1 (table)) suggested the existence of allosteric regulation between monomers at IR-GBSs (although this might not be relevant in vivo , as IR-GBS elements may be occupied by only monomeric GR in the in vivo scenario 26 ). In both cases, DNA acts as a ligand to impart allosteric changes that could affect affinity for co-regulators and ultimately regulatory out-comes, implying that the allosteric transitions extend into the NTD and LBD.…”

Evolutionary model of protein secondary structure capable of revealing new biological relationships

“…We explored three independent studies to demonstrate how well Evolusec (both DSO-sec and JTT-sec) captures evolutionary changes of structural traits. The studies targeted an ancient protein kinase (Wilson et al, 2015), an ancient polar amino acid-binding protein (Clifton and Jackson, 2016), and an ancient DNA-binding domain (Hudson et al, 2016); the proteins represent a range of sequence and structural diversity. Ancestral sequences were inferred from the amino acids of modern proteins, synthesised and their three-dimensional structures were determined.…”

“…Zuckerkandl and Pauling's idea of inferring ancestors from modern proteins (Zuckerkandl and Pauling, 1965) has had recent success in decoding the origins and the determinants of complex protein functions (Hochberg and Thornton, 2017). For example, by comparing inferred ancestors to modern proteins, Wilson et al (2015) elucidated binding affinities in protein kinases, Clifton and Jackson (2016) decoded origins of promiscuity in a solute-binding protein family, and Hudson et al (2016) identified historical amino acid substitutions that alter specificity of a DNA-binding domain. Ancestral sequence reconstruction typically requires a phylogenetic tree, multiple modern protein sequences in an alignment, and an evolutionary model that assigns a probability to evolutionary events.…”