We therefore propose that the conformation of the periplasmic BAY 11-7082 domain generates mechanical strain in BvgS, and that a major function of the PAS domain in BvgS is to maintain, and possibly to amplify, this conformational signal. The complete loss of activity of some BvgS variants generated in this study correlates with strong decreases in

thermal stability of the recombinant PAS domain. The corresponding substitutions thus cause considerably looser structures that most likely make the PASBvg domain unable to maintain and/or transmit the proper conformational strain to the kinase. The importance of the PAS core for stability and activity has also been shown for other PAS domains [35, 36]. Another observation from this and previous work is that a number MI-503 cell line of substitutions in the PAS domain do not inactivate BvgS but render it unresponsive to negative modulation by nicotinate and sulfate selleck [16, 47, 48]. Previously reported substitutions that make BvgS unresponsive to modulation map essentially to a PAS core loop oriented towards the N-terminal flanking helix or to the N-terminal helix itself (Figure 2). It is thus likely that they affect the connection between the PAS core and the upstream region or the stability of the PAS dimer through its N-terminal helices. In the current work, new substitutions that impair or abolish responsiveness to modulation were also identified in the PAS cavity. The

structural stabilities of the latter two PASBvg variant proteins appeared to be decreased to a lower extent than those of the inactive proteins. The observation that the unresponsive BvgS PAS variants remain competent to transmit positive but not negative signals suggests that transmission of modulating signals implies an increased conformational strain relative to the basal, positive-signaling state. Our results do not support the hypothesis that PASBvgS has a heme co-factor. Thus,

the His643Ala substitution does not abolish BvgS activity, as would be expected from the loss of an O2-sensing heme for a strictly aerobic and virulent bacterium. However, this substitution abolishes the response of BvgS to negative Cediranib (AZD2171) modulation, and another substitution in the PAS cavity (Cys607Ala) also decreases BvgS sensitivity to nicotinate. These effects might be explained either by a moderate loosening of the PAS core because the small Ala side chain replaces a larger one, which disrupts the transmission of negative signals, or by a defect in binding a potential intracellular ligand required for transmission of negative signals. The double Tyr596Ala + Asn631Ala substitutions in the PAS cavity that abolish BvgS activity and strongly decrease the PAS thermal stability might also disable ligand binding in vivo. Binding of a cytoplamic ligand by the PAS domain would be consistent with the established link between the nutritional state of B.