STRUCTURAL AND FUNCTIONAL STUDIES OF THE REGULATION AND INHIBITION OF LUXO, A AAA+ ATPASE

Abstract

Quorum sensing is a widespread form of bacterial cell-cell communication that entails the production, release, and detection of small molecules called autoinducers. Autoinducers regulate communal behaviors including biofilm formation and virulence factor production. Here, we focus on LuxO, a shared response regulator protein of the quorum sensing signal transduction pathways in human pathogens including Vibrio cholerae (the bacteria that causes cholera). LuxO is a AAA+ ATPase transcriptional activator. When LuxO is phosphorylated it becomes active, using the energy of ATP hydrolysis to ‘open’ σ54-dependent promoters and thereby upregulate virulence factor production. LuxO inhibitors, identified previously based on high-throughput screening, represent promising leads in the development of novel anti-bacterial drugs that function by modulating quorum sensing.

To elucidate how LuxO is regulated by quorum sensing signals and how inhibitor molecules are able to subvert this regulation, we combined structural, biochemical, and in vivo approaches. The high-resolution X-ray structure of Vibrio angustum LuxO, supported by in vitro and in vivo functional studies, revealed an entirely new mechanism for AAA+ regulation, in which an inter-domain linker occupies the ATPase active site. We also determined the crystal structures of LuxO bound to the broad-spectrum inhibitors AzaU and CV‑133. The inhibitors bind in the ATPase active site and recapitulate elements of the natural regulatory mechanism. Enzymological characterization of two additional LuxO inhibitors, KS12 and ML366, suggests that they function as competitive and uncompetitive inhibitors, respectively. Remarkably, a single molecule of competitive inhibitor (AzaU, CV‑133, or KS12) appears capable of inhibiting an entire LuxO hexamer, implying that ATP hydrolysis is coordinated among subunits. Moreover, initial characterization of mixed hexamers containing both wild-type and ATPase deficient mutant subunits supported the coordination and suggested a partially concerted mechanism for ATP hydrolysis among the subunits of the LuxO hexamer.