Mutations in Sensor 1 and Walker B in the Bovine Papillomavirus E1 Initiator Protein Mimic the Nucleotide-Bound State

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.Viral initiator proteins from DNA viruses belong to the superfamily 3 (SF3) helicases (5, 9). Well-studied members of this group include the T-antigens from the polyomaviruses, the E1 proteins from the papillomaviruses, and the Rep proteins from the adeno-associated viruses. These proteins are multifunctional proteins that utilize ATP binding and hydrolysis by their AAA+ (ATPases associated with various cellular activities) modules for multiple functions in the replication of their viral genomes. AAA+ modules are ∼250-amino-acid ATP binding domains that carry out numerous ATP driven functions (for reviews, see references 6 and 7). For example, the E1 protein, which plays an essential role in papillomavirus DNA replication, has multiple functions that are affected by binding or hydrolysis of ATP (14, 18, 21, 23, 24, 26). E1 is a DNA-binding protein, which binds specifically to E1 binding sites (E1 BS) in the origin of DNA replication (2, 8, 15, 22, 25). DNA-binding activity requires nucleotide binding by E1 (15). In the presence of ATP or ADP, E1 can form a specific double-trimer (DT) complex on the ori and, through ATP hydrolysis, this complex can melt the ori DNA (13, 15, 16). In a process that requires ATP hydrolysis, the DT is then converted into a double hexamer (DH), which has ATP-dependent DNA helicase activity and is the replicative DNA helicase (15, 26). Consequently, the E1 AAA+ module is utilized for ATP binding and hydrolysis in at least two different E1 complexes with different functions. An interesting question is how the same motif for ATP binding and hydrolysis is used in these different complexes to achieve their differing functions.Structural studies of representatives from all three groups—E1 proteins, T antigens, and Rep proteins—have provided important information about how ATP is bound and hydrolyzed by these proteins and the structural consequences that result (1, 4, 7, 10-12). For example, in the recent crystal structure of a hexamer of the E1 oligomerization and helicase domains formed on single-stranded DNA, an ATP binding pocket is formed by 11 residues from two adjacent monomers of the E1 helicase domain (Fig. ​(Fig.1A)1A) (3). Because most of the residues thought to be involved in ATP binding and hydrolysis in these AAA+ proteins are highly conserved and form particular substructures, the specific function of the individual residues have been predicted for these proteins (6) (see Fig. ​Fig.1A).1A). It is well established that the conserved residues in the Walker A and Walker B motifs are involved in both binding and hydrolysis of ATP. The Sensor 1 residues are generally involved in contacting Walker B and the γ-phosphate of ATP. The Sensor 2 motif also participates in nucleotide binding and interacts directly with the γ-phosphate of ATP.Open in a separate windowFIG. 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.To gain a more precise understanding of the role of these particular residues in ATP binding and hydrolysis and because a systematic analysis of such residues has not been performed for the E1 initiator proteins, we performed a mutational analysis of these 11 residues. Based on the behavior of mutants in these residues, the residues can be classified into three groups. Seven of the mutations result in a protein that fails to bind nucleotide and consequently also fail to hydrolyze nucleotide. Three mutants can still bind nucleotide but fail to hydrolyze ATP. Surprisingly, two of these mutants mimic the ATP-bound state and can bind DNA in the absence of nucleotide, demonstrating that E1 utilizes ATP binding for two different modes, only one of which can be mimicked by the mutations in the ATP binding pocket.