We also introduced the Arg670Ala substitution in full-length BvgS, which did not affect its activity in B. pertussis or its ability to respond to negative signals (Figure 4). These observations thus rule out a major function for this residue in PASBvg. More drastic changes in the PAS cavity were next engineered. In the 3BWL structure, the side chains of two Asp residues bind a fortuitously trapped 1H-indole-3 carbaldehyde ligand see more in the PAS cavity. The side chains of the residues at those positions are frequently involved in ligand binding by other PAS domains (our observations), and in the PASBvg cavity these positions are occupied by Tyr596 and Asn631

(Figure 3). They were replaced together by Ala in full-length BvgS. BvgS in the resulting B. pertussis recombinant strain was totally inactive (not shown). We thus verified that BvgSTyr596Ala+Asn631Ala was produced in a stable form in the recombinant B. pertussis strain by preparing membrane extracts and subjecting them to immuno-blotting using polyclonal anti-BvgS antibodies (Figure 5). The protein was detected, showing that the substitutions did not disrupt full-length BvgS or cause its proteolytic degradation but affected its function. selleck Figure 5 Detection of inactive BvgS variants in membrane extracts of the recombinant B. pertussis strains. The immunoblots were revealed

using anti-BvgS STA-9090 polyclonal antibodies. The one-letter code was used to denote the substitutions. ΔbvgS represents BPSMΔbvgS from which bvgS has been deleted. We next determined the effect of the Tyr596Ala + Asn631Ala substitutions on the thermal stability of the recombinant protein. Surprisingly, although N2C3Tyr596Ala+Asn631Ala was purified in a soluble and dimeric form in good amounts, no cooperative denaturation profile was obtained by TSA, and thus no Tm could be calculated. This suggested a significantly looser structure of the PAS core even at lower temperatures. The observations that the joint replacements of Tyr596 and Asn631 in the PASBvg Avelestat (AZD9668) cavity both abolished BvgS activity and considerably destabilized

PASBvg argue that the structural stability of the PAS core domain is important for BvgS function. Of note, mutations in the PAS core have been shown to affect the stability and function of other PAS domains as well [35, 36]. PAS coupling with flanking regions Based on those results, we hypothesized that a major function of the PAS domain is to maintain – and perhaps to amplify- conformational signals coming from the periplasmic moiety of BvgS to the kinase domain, thus requiring a tightly folded PAS core properly connected to the upstream and downstream α helices. To test this hypothesis, we modified residues that couple the PAS domain to its flanking helices and determined the effects of these replacements on BvgS activity.