Tive and focused on V66. However, it is important to recognize that Gc is not zero in any CzrA mutant, consistent with the idea that otherwise non-dominant pathways can make a contribution when a major one is disrupted.54 Our elucidation of what would appear to be a compact pathway of allosteric communication between Zn(II) and DNA binding sites in CzrA stabilized by a cooperative network of van der Waals interactions involving V66 and L68 does not become manifest in a standard pairwise covariation or statistical coupling analysis (SCA) carried out on a large family of ArsR family sensors (Figs. 5), as had been found in previous systems examined by SCA. This is perhaps not so surprising given the involvement of both main chain and side atoms in allosteric hydrogen bonding in CzrA (Fig. 1b), the small subset of interactions that control much of the magnitude of Gc, and the breadth of distinct subfamilies of sensors with different regulatory ligand binding sites and specificities.23 The major finding from this analysis is the identification of a sector of interconnected residues which immediately suggests a way in which the more peripheral elements of the ArsR fold that control DNA binding affinity, i.e., the recognition helices and -wing, move in a concerted fashion (Fig. 6a).Bexarotene The allosteric “hot-spot” identified here centered on V66 and L68 then simply defines a pivot point on which the entire DNA-binding interface is remodeled upon metal binding (Fig. 6b ), rather than an allosteric pathway.56 Rapid evolution of new inducer specificity on this simple scaffold would then become a matter of evolving distinct connectivities to this sector of co-evolving residues (Fig. 6b ), the end result of which is to reposition the DNA-binding levers and/or quench the dynamics in a way that ultimately stabilizes a lowaffinity “open” DNA binding conformation. The recent structures of two ArsR family proteins that are known or projected to exploit thiol-disulfide chemistry to stabilize “closed” and “open” conformations, e.Tropicamide g.PMID:23847952 , B. subtilis HypR24 and Xylella fastidiosa BigR,25 are generally consistent with this model as a common feature that underlies ArsR family protein allosteric function. An alternative view is that this sector comprises a classically defined allosteric network which links the energetically important but more peripheral components of the DNA binding interface, e.g., the N-terminus of the R helix and -wing tips15 to the core of the dimerNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Mol Biol. Author manuscript; available in PMC 2014 April 12.Campanello et al.Pagedefined largely by the 1-5 helical bundle, to achieve a quaternary structural conformational change. Evolution of distinct allosteric sites in the ArsR family would then be possible provided only that these sites physically connect to any point along this sector.54 In this view, the allosteric hot-spot defined here becomes more of an extension of the allosteric ligand binding site itself (Fig. 6b) that mediates a physical connectivity to a preexisting allosteric network. This view is perhaps more consistent with the spectral characteristics of the Zn2-CzrA zrO complex (Fig. 2), which reveal that candidate allosteric residues are interspersed with DNA binding-like residues or alternatively, serve to physically connect the Zn(II) and DNA binding sites on the dimer. The overrepresentation of these residues at the termini of secondary structural elements.
