E0012
DeNovo X-ray Structure Determination of the Bacterial
Quorum-Sensing Phoshorelay Protein Lux U. Jill Dombrauckas1,
Dagny L. Ulrich2, Bernard D. Santarsiero1, Pat
Loria2, Andrew D. Mesecar1, 1Center of
Pharmaceutical Biotechnology, Univ. of Illinois at Chicago; 2Dept. of
Chemistry, Yale Univ.
Lux U is a phosphorelay protein involved in the quorum sensing
circuit of the marine organism Vibrio harveyi. It plays a role in the
pathway that regulates the bioluminescence of the cell. The circuit involves
sensing the population of cells through autoinducer molecules in the
extracellular environment and triggering communication between a series of
proteins that can ultimately suppress or allow transcription of the lux gene.
The structure is of great interest, and will provide valuable information in the
function of Lux U and dynamics in quorum sensing. The sequence of Lux U exhibits
low homology to other two-component response regulator proteins. Therefore, our
approach was to use MAD phasing with the seleno-methionine derivative. Crystals
of the native Lux U were first observed by screening at
the Hauptman-Woodward Institute high-throughput protein crystallization
facility. Final crystallization conditions were determined by optimization of
pH, protein, precipitant, and salt concentrations. Crystals of both native and
seleno-methionine Lux U grew as hexagonal cylinders in space group
P6222 with crystal sizes up to 300x400 microns. X-ray diffraction
data on native and seleno-methionine Lux U were collected at SER-CAT beamline
22-ID at the APS. A three-wavelength MAD data set at the Se-edge was collected
to 2.7Å resolution. SOLVE was used to determine an initial set of phases
to 3.5 Å resolution. Only one selenium site was prominent, and the phasing
power is poor. Preliminary NMR data suggests that Lux U is comprised mainly of
α-helices, and could suggest that the
seleno-methionines are located on the surface of the protein, and thereby
disordered. Other heavy atom derivatives are currently being sought to improve
the initial phases.