Crystal structure of the Vibrio cholerae VqmA–ligand–DNA complex provides insight into ligand-binding mechanisms relevant for drug design

Abstract

VqmA is a highly conserved transcriptional regulator of the quorum-sensing system of Vibrio cholerae, a major human pathogen that continues to imperil human health. VqmA represses biofilm formation and plays an important role in V. cholerae pathogenicity in the human host. Although VqmA's biological function is well understood, the molecular mechanisms by which its specific ligand (and effector), 3,5-dimethylpyrazine-2-ol (DPO), controls transcription of the target gene, vqmR, remain obscure. To elucidate the molecular mechanism of DPO binding, we used structural analyses and biochemical assays to study the V. cholerae VqmA-DPO-DNA complex. These analyses revealed that VqmA contains an N-terminal homodimer domain (PAS) and a C-terminal DNA-binding domain (DBD). We observed that VqmA directly binds to a DPO molecule via a compact hydrophobic pocket, consisting of a sixstranded antiparallel ␤-sheet and several ␣-helices. We also found that the VqmA dimer interacts with the quasi-palindromic sequence of the vqmR promoter through its DBD. The results of the biochemical studies indicated that a water atom and VqmA residues Phe-67 and Lys-101 play a key role in effector recognition, which is also assisted by Tyr-36 and Phe-99. This is the first molecular level view of the VqmA dimer bound to DPO and DNA. The structure-function analyses presented here improve our understanding of the complex mechanisms in the transcriptional regulation of VqmA in Vibrio spp. and may inform the design of drugs to manage V. cholerae infections.

Mechanism underlying autoinducer recognition in theVibrio choleraeDPO-VqmA quorum-sensing pathway

“…S4). This analysis is consistent with the structure of VqmA bound to DNA, which shows that residue K185 makes direct contact with the DNA backbone (17). We compared our structure of DPO-VqmA with a recently reported structure of DPO-VqmA-DNA (17).…”

“…To explore the molecular basis underlying VqmA binding to ligands, we solved the crystal structure of VqmA bound to DPO in the absence of DNA to 2.0 Å using multiwavelength anomalous diffraction (Table S1). The structure of the DPO-VqmA complex bound to DNA was reported recently (17). Additionally, during preparation of this manuscript, the structure of DPO-VqmA without DNA was reported with a similar conformation as in our structure (18).…”

“…We show that the transcriptional activity of VqmA is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae QS transition to HCD upon DPO accumulation. A previous study reported that exogenous addition of DPO to purified Apo-VqmA does not increase the binding affinity of VqmA for PvqmR (17). We find that this is not the case as the DPO-bound VqmA protein has ~2-8-fold higher affinity for DNA than does Apo-VqmA (Fig.…”

“…The VqmA crystal structures reveal that the DPO binding pocket is located in the Nterminal PAS domain. Previous reports compared the VqmA fold to that of LasR, TraR, CviR, and other LuxR-type proteins (17,18), the latter of which, as mentioned, function via simultaneous binding and folding around AHL AIs (13,15,16). While this comparison is certainly informative, it is also worth noting that LuxR-type proteins do not contain PAS domains.…”

“…We solved the crystal structure of VqmA bound to DPO. Comparison to existing structures (17,18) allowed us to provide insight into the conformational changes VqmA undergoes upon DNA binding. We were unable to obtain the crystal structure of VqmA bound to Ala-AA because Ala-AA spontaneously cyclizes into DPO under our crystallization conditions.…”