The Research On ATP-ADP Exchange Activity And Molecular Chaperone Activity Of Hsp70

Heat shock proteins (HSPs), also called stress proteins, are a group of proteins highly conserved in all cells, which are induced when a cell undergoes various types of environmental stresses like heat, cold and oxygen deprivation. Lately it was found they were also present in cells under normal conditions. The 70kDa heat shock proteins (Hsp70s) were the most impotant family in the heat shock proteins. They work as molecular chaperones in eukaryote being involved in various cellular function,such as protein sythesis, folding , translocation, degradation and modulation of protein expression.Now many reseaches are focused on the chaperones mechanism of Hsp70. Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ATP -ADP exchange. Hsp70 exists weakly ATPase activity and locates at the N-teminal 44-kD domain. Recently it was found that besides the activity of ATP hydrolysis Hsp70 exhibited ATP synthesis activity. To identify the function domain of ATP-ADP exchange reaction of Hsp70,we purified the N-teminal 44-kD fragment after extensive digestion withα-chymotrypsin and then characterized the enzyme activity assaying the conversion of [14C]ADP to [14C]ATP and [14C]ATP to [14C]ADP respectively. It has been reported that NDP kinase has weak interaction with Hsp70, which can increase the ATP hydrolysis activity, so we carefully examined the possibility of NDP kinase contamination in the experiment. The equimolar mixture of NDP kinase and Hsp70 were digested and then 44-kD fragment was purified. No different activity was observed for the 44-kD fragment from the mixture of NDP kinase and Hsp70. All the results conformed that N-teminal 44-kD fragment exhibited the intrinsic ATP-ADP exchange activity.During the ATP-ADP exchange reaction Hsp70 forms an acid-labile autophosphorylated intermediate transferingγ- phosphate from ATP to ADP in a similar manner occuring in NDP kinase. In this experiment we analyzed the acid-stable autophosphorylation and its phosphorylated residue of 44-kD fragment. The phosphorylated 44-kD fragment was cleaved with CNBr at Met cleavage sites under acidic condition and 12-kD fragment of Lys128-Met237 showed autophosphorylation after SDS-PAGE and N-terminal amino-acid sequence. Analysis of the digestion of lysyl endopeptidase and N-terminal amino-acid sequence showed that Thr204 was the phosphorylated site which was consistent with the phosphorylation of Thr199 in DnaK.The human Hsp70 and bovine Hsp70 have highly homologic in both sequence and structure. The wide-type and mutant (K71A, T204A and H227S) were expressed in E.Coli and purified on HisTrap^TM Ni²⁺-agarose column and then mono Q anion-exchange column. Lys71,Thr204å’ŒHis227 are important residues in Hsp70 in the two calcium binding sites respectively. No ATP-ADP exchange activity and autophosphorylation were observed in Lys71 and Thr204 mutant and chaperone activity was abolished either. The ATP-ADP exchange activity of H227S point mutation on the second calcium binding site residue wasn't detected but no affect on its phosphorylation and chaperone activity. Moreover ADP partly inhibited the chaperone activity of wide-type Hsp70 but didn't affect the chaperone activity of the mutant on the second calcium binding site residue H227S. The results suggested that the second calcium binding site maybe mainly for ADP binding.