Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus

Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 (similar to75 kDa), encoded by the UL56 and UL89 genes. In a recent investigation, we demonstrated that the main ATPase activity is associated with the large terminase subunit pUL56. The protein has two putative ATP-binding sites, which were suggested to be composed of the sequence (amino acids 463-470) for ATP-binding site 1 and YNETFGKQ (amino acids 709-716) for the second site. We now demonstrate using a 1.5 kb fragment encoding the C-terminal half of pUL56 that ATP-binding site 1 is not critical for the function, whereas ATP-binding site 2 is required for the enzymatic activity. Mutation G714A in this protein reduced the ATPase activity to similar to65% and the double mutation G714A/K715N showed a reduction up to 75%. However, the substitution of E711A revoked the effect of the substitutions. The functional character of the ATP-binding site was demonstrated by transfer of YNETFGKQLSIACLR (709-723) to glutathione-S-transferase (GST). Interestingly, vanadate, an ATPase inhibitor, has the ability to block the ATPase activity of pUL56 as well as of Apyrase, while the antitumor ATP-mimetic agent geldanamycin, did not affect the ATP-binding of pUL56. Furthermore, in contrast to an inactive control compound, the specific HCMV terminase inhibitor BDCRB showed a partial inhibition of the pUL56-specific ATPase activity. Our results clearly demonstrated that (i) the enzymatic activity of the terminase subunit pUL56 could be inhibited by vanadate, (ii) only the ATP-binding site 2 is critical for the pUL56 function and (iii) glycine G714 is an invariant amino acid.