Solution structure and dynamics of Vibrio harveyi LuxU, a phosphotransferase protein involved in bacterial quorum sensing.

摘要

The marine bacterium, Vibrio harveyi controls bioluminescence by a process known as quorum sensing. In this process, autoinducer molecules are detected by membrane bound hybrid sensor kinase proteins (LuxN and LuxQ) that relay the signal through phosphorylation of subsequent proteins in the pathway. The phosphotransferase protein LuxU receives phosphate from both of these proteins, and transfers it to the response regulator protein LuxO. Phosphorylated LuxO indirectly represses the expression of the proteins responsible for bioluminescence. LuxU is of interest because it receives phosphates from two different input sources, and directs them to the same regulatory protein, LuxO. LuxU is a 114 amino-acid residue monomeric protein. Solution NMR was used to determine the first three-dimensional structure of LuxU. The structure was determined from experimental data using CNS, with refinement in explicit solvent using Xplor-NIH. The final refined structure had a backbone RMSD of 0.79 A +/- 0.28 A and a sidechain RMSD of 1.27 A +/- 0.09 A. The structure of LuxU is that of a four-helix bundle, with the four main alpha-helices anti-parallel and twisted about the central axis. This is representative of the family of histidine phosphotransferase proteins. The active site histidine (His58) is located on alpha-helix C with the imidazole sidechain facing into solution. The results of long-range HSQC experiments were consistent with rapid exchange between tautomeric states for His58, and an estimated pKa in the range of 6.0 to 7.0. Two positively charged residues, Lys54 and Lys61, are located near His58 and may participate in stabilization of the phosphate. Aside from these positively charged residues, the binding interface of LuxU is relatively flat and hydrophobic. NMR spin-relaxation experiments identified a collection of flexible residues located in the loop regions of LuxU, which may have a role in conformational changes necessary for interactions with binding partners. The studies described here represent the first structural characterization of an isolated, monomeric bacterial phosphotransferase protein.