In this application Dr. Derewenda proposes to elucidate the structure and function of an acyl-CoA thioesterase III (ACTEIII), an enzyme involved in CD4 down-regulation during HIV-1 infection. One of the key lentivirus proteins required for in vivo infectivity is Nef, a 27 kDa myristoylated molecule. In vitro, Nef induces CD4 down-regulation, enhances infectivity and alters T-cell activation pathways. A novel human protein, ACTEIII, a close homologue of the E. coli thioesterase II (43% amino acid sequence identity) has been found to bind specifically to Nef by the yeast two-hybrid system. The two proteins interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in cells. Further, those nef alleles from human HIV-1 isolates that are unable to down-regulate CD4, either do not react with the thioesterase or they react very poorly. The hACTEIII functions as a mediator of Nef-induced down regulation of CD4. The enzyme's activity against acyl-CoA thioesters has been confirmed in vitro, and it was further found that the association with Nef causes three-fold enhancement of activity. Although the human gene encoding the enzyme has just been discovered its E. coli homologue has been known for some time. Like the human protein, th E. coli TEII is a homotetramer of a total molecular mass of 120 kDa. Unlike other thioesterases, it lacks the typical GXSXG sequence motif that is present in serine hydrolases, and it is not susceptible to serine proteinase inhibitors. Histidine 58 has been implicated in the catalytic mechanism but no details are known. Thioesterases constitute an important family of hydrolytic enzymes involved in many metabolic pathways including fatty acid and polyketid biosynthesis, bacterial luminescence, and regulation of concentration of acyl-CoA derivatives. The ubiquitous modifications of signalling proteins involving myristoylation, palmitoylation and prenylation, suggest that thioesterases are involved in the regulation of protein processing. The thioesterases, which are being investigated in this proposal, could represent larger, hitherto unknown family of hydrolytic enzymes. The investigator proposes to elucidate the crystal structures of the bacterial and human enzyme and to crystallize and determine the structure of the Nef-hACTEIII complex.
