W0189

E.coli Thioesterase Towards Crystal Structure. Jia Li¹, Stuart Smith², Zygmunt Derewenda¹, ¹Department of Molecular Physiology & Biological Physics, University of Virginia, ²Oakland ChildrenÕs Hospital Research Institute, Charlottesville, VA 22908

The E.coli thioesterase II (TEII) is highly homologous with a novel human acyl-CoA thioesterase III (hACTEIII). hACTEIII has been shown to induce CD4 down-regulation in HIV infected cell. This function is mediated by specific binding of hACTEIII to HIV Nef. The tetrameric E.coli thioesterase exhibits very similar catalytic and biophysical properties to that of the hACTEIII, therefore the crystal structure of TEII will lead to better understanding of HIV pathogenesis.

The TEII protein has been overexpressed in a modified pET expression system. The protein can be readily purified from Ni-NTA column. The crystallization condition has been optimized to produce crystals which are suitable for diffraction study. The unit cell parameters are a=94.3_, b=119.9_, c=164.3_, space group C2221. The crystal difracts to 2.8_ at 100K using an R-AXIS IV imaging plate system mounted on a rotating anode. A high resolution (1.9OE) native data set has been collected at EMBL, Hamburg, Germany. The native patterson reveals two peaks and the self-rotation function reveals one peak at kappa=180, phi=90 and omega=45 degree, which indicate that the homotetramer has a 222 non-crystallographic symmetry. One non-crystallographic axis is parrallel to the crystallographic axis c*, while the other two non-crystallographic axes are 45 degree with respect to a* and b* axes. Due to the difficulties in obtaining good heavy atom derivitives, seleno-methionyl TEII has been generated and MAD phasing experiments are underway.