The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2

Abstract

DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.[Keywords: Crystal structure E1E2 complex; papillomavirus; helicase structure; AAA+ protein; DNA replication] Papillomaviruses (PVs) are small DNA viruses that maintain latency in the stem cells of various epithelial tissues (for review, see Howley 1996). They are the etiological agents of a variety of benign lesions of the epithelium, and certain high-risk variants of the human papillomavirus (HPV), such as HPV-16, 18, and 31, are associated with cervical carcinomas (Bosch et al. 1995). The virus is maintained as an extrachromosomal nuclear plasmid that replicates in S phase with the host genome. Because PVs have few open reading frames (∼8 ORFs), the viral life cycle depends heavily on interaction with the host cell for enzymes and ancillary factors for its own gene expression and replication programs. The PVs have provided important novel insights into the regulation of cell proliferation and cancer etiology; for example, the targeted degradation of p53 directed by the HPV16 E6 protein first revealed how key cellular regulatory proteins could be specifically ubiquitinated by E3 ligases for destruction by the proteosome (Scheffner et al. 1993). The PVs also have provided models to understand how eukaryotic transcription factors coassociate to form loops between segments of DNA (Li et al. 1991) and to explore assembly of specific replication factories on defined origins of replication (Stenlund 2003b).The first two universal steps in the formation of a prereplication complex are origin recognition and subsequent distortion and separation of duplex DNA strands. The simplicity of the PV prereplication complex offers advantages for mechanistic and structural dissection of these steps. Only t...

The roles of the residues on the channel β-hairpin and loop structures of simian virus 40 hexameric helicase

“…3B, lanes [7][8][9][10][11][12][13][14][15][16][17][18]. Two other substitutions with long aliphatic side chains, H513M and H513L, also displayed helicase activity (Fig.…”

“…8) (11,(17)(18)(19). However, some AAA ϩ hexameric protein machines, e.g., NSF and p97 for membrane fusion (20)(21)(22), do not possess a similar ␤-hairpin structure.…”

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

“…A prime candidate for the interaction with the TA bp is a ␤-hairpin structure, which is highly conserved in E1 and other papovavirus initiator proteins (1,18). Our previous studies have demonstrated that H507 at the tip of this ␤-hairpin is required for DT formation and for template melting (28).…”

“…Structural studies of E1 and T-Ag have provided important information about the domain structure, how these proteins oligomerize, and how they bind and hydrolyze nucleotides (1,10,13,18). Biochemical analysis has demonstrated that one particular form of E1, a double trimer (DT), generates permanganate reactivity in the ori in the presence of ADP, indicating that the DT provides template melting activity (28).…”

Brd4 links chromatin targeting to HPV transcriptional silencing

“…The full-length E2 protein is a sequence-specific DNAbinding protein implicated in the control of viral DNA replication (Stenlund 2003;Abbate et al 2004), transcription (Hou et al 2002), cell cycle progression (Hwang et al 1993), apoptosis (Blachon et al 2005), senescence (Goodwin and DiMaio 2001), and viral genome maintenance and segregation (Abroi et al 2004;Botchan 2004;McBride et al 2004;Van Tine et al 2004;You et al 2004). Like many cellular transcription factors with different functional modules, E2 contains a C-terminal DNA-binding domain linked to the N-terminal transactivation domain by a flexible hinge.…”