Mutations in Sensor 1 and Walker B in the bovine papillomavirus E1 initiator protein mimic the nucleotide-bound state

Abstract

Viral replication initiator proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ modules for multiple functions in the replication of their viral genomes. These proteins are therefore of particular interest for understanding how AAA+ proteins carry out multiple ATP driven functions. We have performed a comprehensive mutational analysis of the residues involved in ATP binding and hydrolysis in the papillomavirus E1 initiator protein based on the recent structural data. Ten of the eleven residues that were targeted were defective for ATP hydrolysis, and seven of these were also defective for ATP binding. The three mutants that could still bind nucleotide represent the Walker B motif (D478 and D479) and Sensor 1 (N523), three residues that are in close proximity to each other and generally are considered to be involved in ATP hydrolysis. Surprisingly, however, two of these mutants, D478A and N523A, mimicked the nucleotide bound state and were capable of binding DNA in the absence of nucleotide. However, these mutants could not form the E1 double trimer in the absence of nucleotide, demonstrating that there are two qualitatively different consequences of ATP binding by E1, one that can be mimicked by D478A and N523A and one which cannot.

FIG. 1.

(A) Residues in E1 involved in nucleotide binding and hydrolysis. A schematic image is shown of the interface between two E1 monomers that constitute the ATP binding pocket of BPV E1 with the residues that are predicted to be involved in ATP binding and hydrolysis (adapted from reference 3). The Walker A, Walker B, and Sensor 1 and Sensor 2 motifs and the arginine finger are indicated. (B) Formation of the E1₂E2₂-ori complex. EMSA was performed using an 84-bp ori probe. Two quantities (1.5 and 3 ng) of wt E1 and of each E1 substitution, as indicated at the top of the gels, were used in the presence of 0.1 ng of full-length E2. In lane 23, E2 alone was added. The mobility of the E2₂ and E1₂E2₂ complexes are indicated. (C) ATPase activity of E1 substitution mutations of residues involved in ATP binding and hydrolysis. Portions (80 ng) of wt E1 or of each respective E1 substitution mutant as indicated were tested for ATPase activity using ³²P-labeled γ-ATP. After the reaction the free phosphate was separated from ATP by thin-layer chromatography and quantitated by using a Fuji imager. Lane 12 contained [γ-³²P]ATP only.

FIG. 2.

Trimer formation of wt E1 and E1 substitution mutants. wt E1 and the E1 point mutants were tested for complex formation by EMSA on a 39-bp ori probe on which the wt E1 forms a trimer. Three quantities (6, 12, and 24 ng) of wt E1 and the respective E1 mutants were used in the absence or presence of 2 mM ADP as indicated at the top of the panels. (A) D479A and Y534A were tested. (B) D478A and N523A were tested. (C) K425A, K439A, S440A, D497A, Y499A, and R538A were tested in the presence of ADP. (D) R493L, R493M, and R493E were tested in the presence of ADP.

FIG. 3.

E1 DT and DH formation. wt E1 and E1 point mutants were tested for DT and DH formation in EMSA using an 84-bp ori probe. Two quantities of E1 (6 and 12 ng) were used in the absence of nucleotide, in the presence of 2 mM ADP or in the presence of 2 mM ATP as indicated above the lanes. The positions of E1 DT (E1₆) and E1 DH (E1₁₂) are indicated. (A) wt E1, D478A, D479A, and Y534A were tested; (B) wt E1 and N523A were tested.

FIG. 4.

E1 binds ATP with a very short half-life. (A) Schematic description of the experiment shown in Fig. 4B. E1 was incubated in the absence or presence of nucleotide. The samples were then diluted 20-fold. Probe was then added, and the sample then loaded on an EMSA gel. In sample 1 in each set, the binding reaction without ADP was diluted without ADP resulting in a sample without ADP. In sample 2 in each set, the binding reaction was diluted without ADP resulting in a sample containing 0.1 mM ADP. In sample 3 in each set, the binding reaction was diluted with 2 mM ADP resulting in a sample containing 2 mM ADP. (B) wt E1, N523A, and D478A were incubated as described in the scheme in panel A and analyzed by EMSA using the 39-bp trimer probe. Lane 10 contained probe alone.

FIG. 5.

E1DBD interacts physically with the E1 oligomerization and helicase domain. (A) GST pulldown experiments were carried out with the E1DBD GST fusions 142-308 (lanes 4 to 6) and 159-303 (lanes 7 to 9) and a GST fusion of the 60 N-terminal residues of E1 (lanes 2 to 3). The bait was the E1 helicase domain fragment 308-605 labeled by phosphorylation at residue S584 with [γ-³²P]ATP. The pulldown experiments in lanes 2, 4, and 7 were carried out in the absence of nucleotide. The pulldown experiments in lanes 5 and 8 were carried out in the presence of ADP, and the pulldown experiments in lanes 6 and 9 were carried out in the presence of ATP. Lane 1 contains 1% of the input material used in the pulldown experiments. The upper band in the input lane corresponds to autophosphorylated CK2α, while the lower band corresponds to E1 308-605. (B) Diagram illustrating the model for nucleotide dependent DNA binding by E1. In this model the E1 DBD interacts with the E1 helicase domain in the absence of nucleotide, and this interaction prevents DNA binding by the E1 DBD. In the presence of ATP, the nucleotide causes a conformational change in the E1 helicase domain, which results in the release of the E1 DBD, allowing the DBD to bind DNA.