Role of the ATP-binding domain of the human papillomavirus type 11 E1 helicase in E2-dependent binding to the origin

Replication of the genome of human papillomaviruses (BPV) is initiated by t he recruitment of the viral E1 helicase to the origin of DNA replication by the viral E2 protein, which binds specifically to the origin. We determine d, for HPV type 11 (HPV-11), that the C-terminal 296 amino acids of E1 are sufficient for interaction with the transactivation domain of E2 in the yea st two-hybrid system and in vitro. This region of EP encompasses the ATP-bi nding domain. Here we have examined the role of this ATP-binding domain, an d of ATP, on E2-dependent binding of E1 to the origin. Several amino acid s ubstitutions in the phosphate-binding loop (P loop), which is implicated in binding the triphosphate moiety of ATP, abolished E2 binding, indicating t hat the structural integrity of this domain is essential for the interactio n. The structural constraints imposed on the E1 P loop may differ between H PV-11 and bovine papillomavirus type 1 (BPV-1), since the P479S substitutio n that inactivates BPV-1 E1 is tolerated in the HPV-11 enzyme. Other substi tutions in the E1 P loop, or in two other conserved moths of the ATP-bindin g domain, were tolerated, indicating that ATP binding is not essential for interaction with E2. Nevertheless, ATP-Mg stimulated the E2-dependent bindi ng of E1 to the origin in vitro. This stimulation was maximal at the physio logical temperature (37 degrees C) and did not require ATP hydrolysis. In c ontrast, ATP-Mg did not stimulate the E2-dependent binding to the origin of an E1 protein containing only the C-terminal domain (353 to 649) or that o f mutant E1 proteins with alterations in the DNA-binding domain. These resu lts are discussed in light of a model in which the E1 ATP-binding domain is required for formation of the E2-binding surface and can, upon the binding of ATP, facilitate and/or stabilize the interaction of E1 with the origin.