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Crystal Structure of the SF3 Helicase from Adeno-Associated Virus Type 2

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Crystal Structure of the SF3 Helicase from Adeno-Associated Virus Type 2 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Structure, Vol. 11, 1025–1035, August, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0969-2126(03)00152-7 Crystal Structure of the SF3 Helicase from Adeno-Associated Virus Type 2 J. Anson James,1,3 Carlos R. Escalante,2,3 AAV establishes a latent infection by site-specifically Miran Yoon-Robarts,1 Thomas A. Edwards,2 integrating its genome into a locus on human chromo- R. Michael Linden,1,* and Aneel K. Aggarwal2,* some 19 (19q13.4), termed AAVS1 (Kotin et al., 1990; 1 Carl C. Icahn Center for Gene Therapy Samulski et al., 1991; Dutheil et al., 2000). This ability and Molecular Medicine to establish latency through targeted integration is an 2 Department of Physiology and Biophysics aspect of the AAV lifecycle that is unique among mam- Mount Sinai School of Medicine malian viruses. To date, AAV infection has not been 1425 Madison Avenue associated with any apparent pathologies, making it New York, New York 10029 an attractive vector for human gene therapy. This is especially notable in light of the fact that AAV infection is widespread; over 80% of the human population is Summary estimated to be seropositive for anti-AAV antibodies (for a review, see Linden and Berns, 2000 and references We report here the crystal structure of an SF3 DNA therein). helicase, Rep40, from adeno-associated virus 2 (AAV2). The AAV genome is a 4.7 kb single-stranded DNA We show that AAV2 Rep40 is structurally more similar molecule that consists of two open reading frames to the AAA؉ class of cellular proteins than to DNA (ORFs), rep and cap. Together, the ORFs encode four helicases from other superfamilies. The structure de- nonstructural and three capsid proteins, respectively. lineates the expected Walker A and B motifs, but also Consistent with their multifunctional role during the viral reveals an unexpected “arginine finger” that directly lifecycle, the products of the rep ORF, Rep78, -68, -52, implies the requirement of Rep40 oligomerization for and -40 (designated by their apparent molecular weight), ATP hydrolysis and helicase activity. Further, the possess a variety of biochemical activities (Im and Mu- Rep40 AAA؉ domain is novel in that it is unimodular zyczka, 1990). The three major functional domains are as opposed to bimodular. Altogether, the structural all present in the largest of the Rep proteins, Rep78. -connection to AAA؉ proteins defines the general archi- The Rep amino terminus possesses specific DNA bind tecture of SF3 DNA helicases, a family that includes ing and endonuclease activity, the central domain bears simian virus 40 (SV40) T antigen, as well as provides motifs necessary for ATPase and helicase activity as a conceptual framework for understanding the role of well as the nuclear localization sequence, and the car- Rep proteins during AAV DNA replication, packaging, boxy-terminal Zn finger domain has been implicated in and site-specific integration. interacting with a myriad of cellular factors. The re- maining Rep isoforms, Rep68, -52, and -40, are a combi- Introduction nation of these functional domains arising from alterna- tive splicing schemes and differential promoter usage Small DNA viruses have been widely exploited to study within the rep ORF. Notably, all four isoforms possess cellular processes such as DNA replication and tran- the helicase domain, with Rep40 representing the mini- scription, protein translation, and, in several instances, mum AAV helicase (see Figure 1A). The majority of bio- tumor transformation. Their typically simple genome chemical studies focus on the ability of the Rep proteins structures encode proteins that essentially function to to mediate viral origin-dependent DNA replication and (1) orchestrate cellular factors to provide a suitable envi- site-specific integration. However, the only structural ronment for the virus lifecycle and (2) provide the neces- information available to date on Rep proteins is a crystal sary factors for viral replication, gene expression, and structure of the Rep origin interaction domain of human particle assembly. Consequently, the nonstructural pro- AAV serotype 5 (Hickman et al., 2002). The lack of struc- teins of these viruses have evolved to be multifunctional tural information has limited the molecular assessment with respect to their inherent biochemical activities. Ad- of the AAV lifecycle, including the assembly pathways eno-associated virus type 2 (AAV2), a human parvovirus, attributed to ATPase and helicase activities (King et al., is a representative member of such DNA viruses. 2001), as well as the mechanism underlying site-specific First identified as a contaminant of adenoviral stocks, integration. the name adeno-associated virus reflects the require- DNA helicases have been found to be involved in a ment of a helper virus coinfection, typically adenovirus, range of cellular processes, including DNA replication, in order for AAV to undergo productive replication. How- repair, transcription, translation, and viral packaging (re- ever, it is now widely accepted that concomitant infec- viewed by Lohman and Bjornson, 1996). Typically, the tion by other viruses, as well as particular cellular states, energy required for the helicase-mediated unwinding of can provide helper functions in support of productive DNA is provided by NTP hydrolysis. Helicases possess AAV replication. In the absence of favorable cellular con- a number of conserved motifs necessary for this ATPase ditions for productive replication (i.e., in healthy cells), function and translocation along the DNA/RNA mole- cule. A general classification scheme was proposed by *Correspondence: [email protected] (R.M.L.), aggarwal@ Gorbalenya and Koonin (1993) based on the organiza- inka.mssm.edu (A.K.A.) tion of these functional motifs. As a result, five major 3 These authors contributed equally to this work. superfamilies were identified, designated SF1–SF5. The Structure 1026 Figure 1. Schematic Representation of the AAV2 Rep Proteins (A) A schematic diagram of the AAV2 Rep proteins, with N-terminal origin interaction domain (red), helicase domain (gold), and Zn finger domain (green). Dashed rectangles indicate Walker A, Walker B, and motif C. The position of the arrow shows the truncated form (Rep40⌬490) used for crystallization. (B) Helicase assay. Rep40, Rep40⌬490, and Rep40⌬490 bearing a mutation of the Walker A lysine (K340H) were titrated at various concentrations on partially duplex M13 DNA substrates. Helicase assays were performed as described in Experimental Procedures. The amount of each Rep variant is indicated in pmoles. S, substrate; P, displaced single-stranded product; Ϫ, no protein; boil, heat-denatured substrate. (C) Structure-based sequence alignment of the helicase domains of representative members of the SF3 family. Shown above the alignment is the relative location of ␣ helices and ␤ strands in the Rep40 structure; the N-terminal domain is colored blue, and the C-terminal ␣/␤ domain is colored gold. Boxed regions correspond to the SF3 signature motifs. The arginine finger residue is marked with an asterisk. Included in the comparison are: AAV2_Rep, adeno-associated virus 2 Rep40 protein; PPAV_NS1, porcine parvovirus NS1 protein; SV40_TAg, simian virus 40 large T antigen; MPOV, mouse polyomavirus; HPV1a_E1 human papillomavirus type 1a E1 protein; BPV1_E1 bovine papillomavirus type 1 E1 protein. Green, lightly conserved regions; brown, absolutely conserved regions. AAV Rep helicase falls into the SF3 class, as do many coordination site, and sensor 1 site, respectively. SF3 viral helicases including that of papillomaviruses, polio- helicases have been shown to form hexamers or double virus, and simian virus 40 (SV40). Members of the SF3 hexamers, and exhibit 3Ј ⇒ 5Ј NTP-dependent helicase family contain four highly conserved regions within a activity. short stretch of approximately 100 amino acids: motifs While much is known about the biological activity of A, B (also known as Walker motifs A and B), BЈ, and C several SF3 helicases (particularly SV40 T antigen), a essentially make up the core of the helicase active site detailed assessment of the DNA unwinding mechanism consisting of an NTP binding site, divalent metal cation by SF3 helicases has been hindered by the lack of high- Crystal Structure of the SF3 Helicase from AAV2 1027 resolution structural data of a representative from this more common “triangular”) look to the Rep40 AAAϩ group. Here, we present the high-resolution structure domain. We will refer to this new domain as vAAAϩ, for of AAV2 Rep40, an SF3 helicase. We find that Rep40 is viral AAAϩ, to differentiate it from the “classical” AAAϩ structurally related to the AAAϩ (ATPases associated domain. with diverse cellular activities extended family) of cellu- lar ATPases, and find that Rep40 possesses a novel AAAϩ module predicted to be shared by other viral SF3 Rep40, an SF3 Helicase, Defines ؉ helicases. a Novel AAA Architecture The closest structural homologs to Rep40 are AAAϩ proteins. Representative members of AAAϩ proteins in- Results and Discussion clude the motor protein RuvB, responsible for Holliday junction migration (Putman et al., 2001; Yamada et al., Structure Determination 2001); N-ethylmaleimide-sensitive fusion protein (NSF- We determined the structure using the multiwavelength D2), which plays a role in membrane fusion (Lenzen et anomalous diffraction (MAD) method (Hendrickson, al.,
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