Our x-ray structural studies demonstrate that CviR and CviR′ can each populate closed conformations that lock the DNA recognition helices into an arrangement incompatible with operator DNA binding (). The most stable of these closed conformations, by all of our criteria, is the CviR:CL complex, in which readjustments in the LBD () allow it to interact favorably with the DBD in a crossed-domain interaction. A distinct crossed-domain conformation is adopted by CviR′:C6-HSL, in which a cavity generated by the absence of four carbons present in the native ten-carbon autoinducer makes room for the side chain of a Met residue on the DBD (). The closed conformation adopted by CviR′:C6-HSL provides independent validation for the strategy of inter-domain stabilization. Strong additional support comes from our finding that, for CviR′, mutations that convert C10-HSL from an agonist to an antagonist map to the LBD-DBD interface near the ligand-binding site (residues 77 and 89; see ). Our results are consistent with previous reports for other LuxR-type proteins, which demonstrate that relatively minor changes in ligand structure can have drastic effects on agonist/antagonist activity (Geske et al., 2007
; Geske et al., 2005
; Müh et al., 2006
). Indeed, we find that large changes in activity can result from subtle conformational rearrangements of the receptors caused, in turn, either by alterations in the ligand or by amino acid changes in the protein.
Relative to the CviR:antagonist complexes we studied, we find that CviR:agonist complexes are more susceptible to in vitro proteolytic cleavage (), possibly because – unlike the antagonist-bound complexes – they are dynamic and occupy an ensemble of conformations. Indeed, flexibility in the LBD-DBD connection may be important for DNA engagement, based on the highly asymmetric structure of the CviR homolog TraR bound to operator DNA (Vannini et al., 2002
; Zhang et al., 2002
). Neither the intrinsic flexibility of CviR:agonist complexes nor the extent to which they populate crossed-domain conformations has been examined directly. It nevertheless seems likely based on Cys cross-linking experiments (Figure S2
) that the antagonist complex structures capture crossed-domain conformations only fleetingly present in agonist complexes, simultaneously rendering them inactive for DNA binding and suitable for crystallization.
Many proteins contain domains connected by flexible linkers. Their physical connection means that these domains are maintained at a high effective concentration with respect to one another. Here we show that it is possible to develop antagonists that function by stabilizing transient inter-domain interactions, giving rise to an inactive configuration. For quorum-sensing receptors of the LuxR family, there are two general strategies by which one might accomplish this goal. The first strategy, demonstrated here, is to identify ligands that bind in place of the autoinducer and that – to a greater extent than the autoinducer – favor an inactivating LBD-DBD interaction. A second, related approach toward antagonist development would be to identify molecules that bind to pre-formed LuxR-autoinducer complexes in such a way as to stabilize closed conformations. No examples of this latter type of small-molecule antagonist have to our knowledge been reported. The natural quorum-sensing inhibitor protein TraM, however, employs a related mechanism, inserting itself into TraR:autoinducer complexes between the ligand-binding and DNA-binding domains such that the latter are mis-positioned for DNA binding (Chen et al., 2007
). This inactive TraR configuration bears no resemblance to the crossed-domain configuration characterized here for CviR, indicating that diverse DNA-binding deficient conformations might in principle be stabilized by different natural or synthetic antagonists. In practice it seems likely that most therapeutics will stabilize transient configurations normally adopted, albeit with much lower probability, by agonist-bound LuxR proteins. Strategies aimed at stabilizing transient interdomain interactions thus appear particularly promising for antagonist design.
The canonical LuxR-type protein binds AHL, which induces protein dimerization, DNA binding, and RNA polymerase activation. A second class of LuxR-type receptor is exemplified by EsaR, which folds, dimerizes, and binds DNA in the absence of its AHL ligand (Minogue et al., 2002
; von Bodman et al., 2003
). DNA binding by apo-EsaR results in transcriptional repression of quorum sensing regulated genes. This repression is alleviated by the binding of the native autoinducer to EsaR, which inhibits EsaR’s DNA binding activity (Minogue et al., 2005
). Based on our results, we speculate that autoinducer binding to EsaR may stabilize a crossed-domain conformation unable to bind DNA. This model could thereby explain the mechanism underlying the regulation of this second class of LuxR-type proteins.