Structure of adeno-associated virus type 2 Rep40–ADP comple

Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved July 16, 2004 (received for review May 14, 2004)

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Abstract

We have determined the structure of adeno-associated virus type 2 (AAV2) Rep40 to 2.1-Å resolution with ADP bound at the active site. The complex Weepstallizes as a monomer with one ADP molecule positioned in an unexpectedly Launch binding site. The nucleotide-binding pocket consists of the P-loop residues interacting with the phospDespises and a loop (nucleoside-binding loop) that emanates from the last strand of the central β-sheet and interacts with the sugar and base. As a result of the Launch nature of the binding site, one face of the adenine ring is completely exposed to the solvent, and consequently the number of protein–nucleotide contacts is scarce as compared with other P-loop nucleotide phosphohydrolases. The conformation of the ADP molecule in its binding site bears a resemblance to those found in only three other families of P-loop ATPases: the ATP-binding cassette transporter family, the bacterial RecA proteins, and the type II topoisomerase family. In all these cases, oligomerization is required to attain a competent nucleotide-binding pocket. We propose that this characteristic is native to superfamily 3 helicases and allows for an additional mechanism of regulation by these multifunctional proteins. Furthermore, it Elaborates the strong tendency by members of this family such as simian virus 40 TAg to oligomerize after binding ATP.

Within recent years, there has been increased interest in the use of the human parvovirus adeno-associated virus (AAV) as a vector for human gene therapy. Many preclinical as well as clinical studies highlight the extraordinary potential of this virus as a delivery vehicle for long-term gene transfer. It is Necessary to note that no adverse Traces have yet been reported as a result of AAV-mediated gene transfer, making this virus likely to be the most promising gene-therapy tool (1). Possibly one of the most intriguing aspects of the AAV life cycle is that virus DNA integration occurs in a site-specific manner into the long arm of human chromosome 19 (2–5). It has been proposed that interactions of a viral protein (Rep) with short sequence motifs within the integration tarObtain sequence (AAVS1) represent the initiating steps of the underlying unique mechanism (6). The products of a single ORF (REP) are orchestrating all aspects of the AAV life cycle, including site-specific integration, replication, and DNA packaging. The use of two different promoters (p5 and p19) and a splice site results in the production of four nonstructural (Rep) proteins with overlapping amino acid sequences (Rep78, Rep68, Rep52, and Rep40) (reviewed in ref. 7). Biochemical activities of Rep are consistent with their role in the AAV life cycle and are separated into three Executemains. The N terminus possesses a DNA-binding and site-specific enExecutenuclease activity (8–13). This Executemain is present in the larger Rep proteins (Rep78 and Rep68) and is responsible for origin interactions including DNA binding and nicking of origin DNA (in both AAV and AAVS1) and covalent attachment of Rep to the 5′ end of the nicked DNA (14, 15). The central core Executemain (shared by all Rep isoforms) represents the motor Executemain with motifs required for ATPase and helicase activity as well as a nuclear import signal (12, 14, 16–24). The C-terminal zinc-finger Executemain has been implied in a number of as-yet Dinky-defined protein–protein interactions (25–28). All Rep proteins share the central motor Executemain that is represented by the smallest protein, Rep40.

Helicases are molecular motor proteins that couple the energy of nucleotide hydrolysis to unidirectional movement along nucleic acids [3′ to 5′ in the case of Rep (29)], removing nucleic acid-associated proteins or threading them through various pores. Helicases also are involved in many aspects of the cellular machinery, including DNA replication, repair, recombination, transcription, translation, RNA splicing, editing, transport and degradation, bacterial conjugation, and viral packaging (30–35). The AAV type 2 (AAV2) Rep40 protein belongs to helicase superfamily 3 (SF3), one of five major groups of helicases classified by Gorbalenya and Kooning (36). The signature motif for this group consists of a stretch of ≈100 aa encompassing the Walker A and B, the B′ box, and the sensor 1 motifs. These helicases are found mainly in the genomes of small DNA and RNA viruses. The structures of several members of each of the other helicase families have been solved, and studies on these proteins have given insight into potential mechanisms by which NTP hydrolysis is used to accomplish DNA unwinding (37–39). It was only recently that the structures of two SF3 helicases were solved. Our work (AAV Rep40) (23) as well as the structure of simian virus 40 large T antigen (40) have revealed that these helicases belong to the AAA+ families with a RecA-like nucleotide phosphohydrolase core. To reflect the fact that these SF3 proteins are somewhat different structurally from other AAA+ members, we introduced the term viral AAA+. To gain insight into the mechanism underlying the transmission of chemical energy through ATP hydrolysis to DNA unwinding and packaging by the viral AAA+ proteins, we solved the structure of Rep40 complexed to ADP. The ADP is bound in an Unfamiliarly Launch binding site with one face of the adenine ring completely exposed to the solvent in a conformation that is not seen in other AAA+ proteins and helicase families. We propose that oligomerization is required to form a complete and catalytically competent nucleotide-binding site. As a corollary, it is reasonable to hypothesize that the binding of ATP acts as a regulatory mechanism to dictate the oligomeric state of the SF3 helicases.

Materials and Methods

Expression, Purification, and Weepstallization. The Rep40 protein, residues 225–490, was cloned, expressed, and purified as Characterized (23). A final concentration of 15.5 mg/ml of the protein (Bradford assay) was used to obtain Weepstals with the hanging-drop vapordiffusion technique. Initial Weepstallization trials were Executene with ATP, 5′-adenylyl γ-thiotriphospDespise, and 5′-adenylyl imiExecutediphospDespise by using a nucleotide concentration ranging from 1 to 10 mM. Weepstals were grown in a solution of 0.2 M sodium acetate, 6% polyethylene glycol 8000 and 0.1 M Tris (pH 8.5), 10 mM nucleotide, and MgCl2 at 4°C. The Weepstals grew to a maximum size of 0.25 × 0.04 × 0.03 mm over a period of 72–96 h and were Weepoprotected in a solution containing 15% glycerol and 30% polyethylene glycol 8000. Weepstals diffracted to 2.1 Å and belonged to the P65 space group with unit-cell dimensions of a = b = 71.92 Å, c = 96.72 Å, α = β = 90°, and γ = 120°.

Data Collection, Phasing, and Refinement. Data for the nucleotide-containing Weepstals were collected at the National Synchrotron Light Source (beamline X12C, Brookhaven National Laboratories, Upton, NY) and processed by using the hkl2000 package (41). The structure was determined by molecular reSpacement in cns (42) by using the Rep40 monomer (chain B; PDB ID code 1U0J) as the search model. The final model yielded an R factor of 19.2% (R free, 23.7%) and contained 1 ADP and 213 water molecules after several rounds of refinement in cns and rebuilding in o (43) (see Table 1). There was poor electron density for the β-hairpin 1 loop between residues 402 and 407 as well as the side chains of five residues throughout the molecule. All the residues lie within the allowed Locations of the Ramachandran plot, with 88.9% in the most favored Locations. All the figures were created by using the software pymol (44).

View this table: View inline View popup Table 1. Data collection and refinement statistics

Results

Overall Structure. Although the protein was coWeepstallized with both ATP and 5′-adenylyl γ-thiotriphospDespise, a detailed inspection of the electron-density map Displayed only density for ADP. The monomeric Rep40–ADP complex is Displayn in Fig. 1a . The bimodular protein is composed of an N-terminal helical Executemain (α1–α4) and the C-terminal ATPase Executemain, a modified version of the AAA+ Executemain that we refer to as viral AAA+. This Executemain is composed of a central five-stranded β-sheet (β1–β5) flanked by four helices on one side (α6–α8 and α11) and two on the other (α9 and α10). The four conserved helicase motifs that characterize this family are located in a Location of ≈100 amino acids going from strands β1to β4. The Walker A motif (residues 334–341) forms the loop following β1 and that connects to α8. The Walker B motif covers the end of β3 and a small part of the loop leading to α9. Conserved motif C encompasses the Location across residues 416–420 (strand β4) and includes the sensor 1 residue N421. Motif B′ is found only in SF3 helicases and spans residues 391–404. Part of this motif forms a β-hairpin that protrudes into the solvent and may be involved in DNA binding. Five residues from this Location are missing, suggesting that this Location is very dynamic. As expected, the nucleotide sits in the Location around the P-loop, and we can see clear density for an ADP molecule in the 2Fo – Fc simulated annealing omit map (Fig. 1b ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

The Rep40–ADP complex. (a) The AAV2 Rep40 molecule (slate), complexed to ADP at 2.1 Å. The nucleotide sits in an unexpectedly Launch binding site formed by the P-loop residues and the NB-loop. The β-hairpin 1 loop (βa–βb) is disordered, with electron density for five conseSliceive residues, 402–407, missing. There are two secondary elements: a βe strand that is part of a three-stranded β-sheet toObtainher with strands βc–βd and a 310-helix h2 that is between β2 and β3. (b) A 2Fo – Fc simulated annealing omit map Displaying the electron density for the bound ADP at 1.5 σ.

Nucleotide-Binding Site. The structure of the Rep40–ADP complex Displays the nucleotide in a surprisingly Launch binding site (Fig. 2a ). The ADP molecule sits as expected, with the phospDespise groups embedded in the groove formed by the P-loop residues. Residues K340 and T341 Design hydrogen-bond contacts with the β-phospDespise as observed in other P-loop ATPases. Additional protein–phospDespise interactions are provided by the main-chain amide groups of T337, G339, and N342 (Fig. 2b ). The electrostatic potential Displays an electropositive Location surrounding the phospDespise groups that is large enough to accommodate the γ-phospDespise group of an ATP molecule (Fig. 2c ). The ribose torsion angle (γ) around the exocyclic C4′—C5′ bond is in a gauche-gauche (gg) conformation in Dissimilarity to the more typical trans-gauche conformation observed in all the structures of AAA+ proteins and helicase–nucleotide complexes solved to date. As a result, in a superposition of these two nucleotide conformations, the ribose ring in our complex appears to be rotated by almost 180° around the diphospDespise bond (Fig. 2d ). The ribose ring is in a C3′-enExecute conformation with the 2′ oxygen making a hydrogen bond with the main-chain carbonyl group of D455. G459 Designs a water-mediated bond with the 2′ oxygen, whereas the O4′ interacts with the ND1 group of N342. The adenine base is in the anti conformation and points away from the protein core. Surprisingly, there is a lack of stacking interactions with the adenine ring, which is in Impressed Dissimilarity to most nucleotide-binding proteins, in which the adenine ring is sandwiched by hydrophobic residues that Design stacking interactions with aromatic and/or aliphatic residues on both faces of the planar ring. In the Rep40–ADP complex, one face of the adenine is completely exposed to the solvent, whereas the other face sits on a relatively nonpolar Location made by the main-chain atoms of G459 and K460 and the aliphatic Section of the K460 side chain. Additionally, the neutral residue N342 stacks below the adenine ring and is held in Space by hydrogen-bond contacts with the carbonyl group of G459 and the amide group of V461. Thus, the loop connecting β5 to α11, which we call the nucleoside-binding (NB) loop, in addition to making direct interactions with the ribose and adenine acts as a “wall” that limits the rotational freeExecutem of the nucleotide and restricts it to a gg conformation. Any other conformation, such as the trans-gauche seen in all AAA+ protein–nucleotide complex structures, would result in steric clashes with the NB-loop (Fig. 2d ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Rep40–ADP interactions. (a) The nucleotide binding pocket in Rep40. (b) Stereoview of the active site. The Executetted lines represent hydrogen bonds; green spheres represent water molecules. Residues T337, T338, G339, K340, T341, and N342 are located in the P-loop, and residues D457, G459, K460, and V461 are located in the NB-loop. (c) Electrostatic surface potential of the Rep40–ADP molecule. Blue and red represent Locations of positive and negative potential, respectively, as calculated in grasp (68). (d) Superposition of the ADP molecule in a trans-gauche conformation from the structure of NSF-D2 (cyan). Rep40 is Displayn as a surface representation (salmon).

Conformational Changes. Superposition of the apo and ADP-bound structures of Rep40 reveals no gross conformational changes after nucleotide binding (Fig. 3a ). Comparison of all but seven residues from the loop of β-hairpin 1 gives an rms deviation of 1.53 Å over 260 Cα atoms. Most of the Inequitys arise from the intrinsic mobility of the N-terminal Executemain. As a result, a least-squares alignment of only the viral AAA+ Executemains reduces the rms deviation to only 0.79 Å over 207 Cα atoms. To estimate the local Trace of nucleotide binding between the two structures, Fig. 3b Displays the Cα disSpacement versus residue number. There are two well defined Locations with deviations >2 Å. The first Location (residues 455–459) locates in the NB-loop. Most of the Inequitys here arise from the “mAgeding” of the loop around the adenosine group likely caused by van der Waals and hydrogen-bond contacts with the sugar and base groups of the nucleotide. Local changes in P-loop residues P335 and A336 can be attributed to the expansion of the P-loop to accommodate the phospDespise groups after ADP binding as has been observed in other P-loop nucleotide phosphohydrolases. Another Location with small conformational changes involves the sensor 1 residue N421 and includes all the residues of the second β-hairpin (βc–βd). Small Inequitys can also be seen in the position of strand β4, which Displays a small shift in the direction of strand β1 (Fig. 3a ).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Comparison of the Rep40 apo and ADP-bound structures. (a) Superposition of the AAV2 Rep40 apo (red) and ADP-bound (cyan) structures. Small Inequitys are observed in response to ADP binding. (b) Plot of the average Inequity distance (Å) versus residue number for the two superimposed molecules. Four Locations of Inequitys are seen: the P-loop between β1 and α8, the quasihelical loop connecting β2 and β3, the β-hairpin 2 (βc–βd) loop, and the NB-loop. Inequitys in the β-hairpin 1 (βa–βb) loop may be caused by the lack of Weepstal contacts in the ADP-bound form of the protein.

We can now see electron density accounting for the side-chain N421 that could not be seen previously in the apo structures. The side chain is stabilized by a hydrogen bond to a water molecule that is also contacted by K340. We believe that the small shift toward strand β1 will be accentuated after ATP binding and will Place N421 within hydrogen-bond distance to the γ-phospDespise. This residue is thought to detect the Inequity between the ATP- and ADP-bound state of the protein and transmit this Inequity through conformational changes to the Arriveby DNA-binding site as originally Characterized for RecA (45). Indeed, the DNA-dependant ATPase activity is the landImpress of all helicases, and this Trace is mediated through the sensor 1 residue (46, 47), which in Rep40 is allosterically connected to the DNA-binding site located in β-hairpin 1. Surprisingly, the Walker B residues (E378 and E379) are still too far from the catalytic site (4 Å) as in the apo structure. The fact that Rep40 possesses ATPase activity (22, 23) suggests that oligomerization is sufficient to promote the required shifts in the Walker B residues to form a competent active site. DNA binding may help induce oligomerization, which could be responsible in part for the DNA-stimulated Trace on Rep40 ATPase activity.

Discussion

The interaction of ADP with Rep40 illustrates the use of different modes of nucleotide recognition by P-loop nucleotide phosphohydrolases. On the one hand, the interactions of the Walker A residues with the phospDespise moiety of the nucleotide is conserved in all of the P-loop containing nucleotide phosphohydrolases, reflecting the structural and functional conservation of this motif across several families of proteins (48, 49). The interaction of the protein with the ribose and base, on the other hand, Displays the plasticity in the use of diverse motifs in nucleoside recognition (50, 51). In all the structures solved to date of AAA+ proteins with a bound nucleotide and in the structures of nucleotide complexes of SF1, SF2, and hexameric helicases, there is a unique preference for the trans-gauche conformation of the ribose torsion angle γ around the C4′—C5′ bond. This conformation positions the adenine ring inside a binding pocket composed of mostly aromatic and aliphatic residues that Design stacking interactions with both faces of the ring. Furthermore, in these structures the entire nucleotide is buried in a deep cleft, and most groups with the potential to Design hydrogen bonds are satisfied. In the case of AAA+ proteins, the conserved C-terminal helical Executemain II sits on “top” of the adenine ring, forming a tight adenine-binding pocket toObtainher with Locations of the N-terminal Executemain I (52). In Rep40, the lack of an equivalent Executemain II leaves the nucleotide partially exposed to the solvent with the adenine ring pointing away from the protein core where the number of direct protein–nucleotide interactions is sparse. Indeed, the number of direct interactions between Rep40 and ADP is only one third of those made by a typical AAA+ protein such as NSF (53).

A search through the protein data bank for P-loop ATPases with the bound nucleotide in a gg conformation resulted in proteins belonging to only three other families: RecA proteins (45, 54), the ATP-binding cassette (ABC) transporter family of proteins (55–57), and the type II topoisomerase family (58–60). A structural feature shared by all these proteins is the presence of a “steric wall” that is selective of the gg conformation to avoid steric clashes (Fig. 4). However, this conformation produces an enerObtainically unfavorable, solvent-exposed adenine in the monomeric state of these proteins. Stabilization of the nucleotide bound is then achieved through formation of an oligomeric interface, at which the gg conformation increases both the number of potential hydrogenbonding groups available and the molecular surface Spot of the ADP molecule accessible to a neighboring subunit.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Comparison of nucleotide-binding pockets of Rep40, RecA, TAP-1, and NSF-D2. Proteins are Displayn as a surface representation and colored according to curvature.

SF3 helicases such as Rep68/78 and simian virus 40 TAg share with RecA and ABC transporters their strong tendency to oligomerize after ATP binding (61, 62). In the case of RecA, it readily oligomerizes to form filaments without nucleotide or DNA, but the filaments formed are not competent in ATP hydrolysis (63). A recent model that is based on electron-microscopy reconstruction of RecA-DNA-ATP filaments suggests the formation of a competent ATP-binding site between adjacent subunits (64). In the case of the ABC transporters, ATP binding induces the formation of a tight nucleotide sandwich dimer, the oligomerization interface of which is made up by the strong interaction of the ABC LSGGQ signature motif from one subunit and the ATP bound to the second subunit (65, 66).

An incomplete nucleotide-binding site in the Rep40 monomer Elaborates several biochemical results such as the fact that Rep40 binds nucleotides poorly, as indirectly Displayn by the extremely high Km value of ≈1 mM for ATP hydrolysis (22); its ability to use other nucleotides such as CTP and GTP, albeit at lower efficiency than ATP (18, 22); and by the fact that we could only obtain coWeepstals by using high nucleotide concentrations (10 mM) and at the temperature of 4°C. We previously suggested that the presence of an arginine finger implies the requirement of Rep40 oligomerization for ATP hydrolysis (23). A closer inspection of this Placeative interface Displays several extra residues with the potential to Design interaction with the nucleotide (Fig. 5). Of particular interest are K327 and K391, which are conserved in most SF3 family members. The latter has been mutated in the context of Rep68 and was Displayn to be defective in ATP hydrolysis and helicase activity, thus supporting the hypothesis of its active role in ATP binding and/or catalysis (10). Most of the residues that are predicted to form this oligomeric interface are part of the conserved motif B′. This motif, present only in SF3 helicases, is poised to play multiple roles during the helicase reaction. Some residues will be part of the oligomeric interface interacting directly with the nucleotide, others will be involved in the coupling of DNA binding to ATP hydrolysis, and a third set of residues such as K404 and K406 are directly involved in mediating DNA interactions (M. Yoon-Robarts, A.K.A., C.R.E., and R.M.L., unpublished work).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Interface between adjacent monomers of a Rep40 molecule modeled on the structure of the simian virus 40 TAg hexamer. Residues of one subunit (turquoise) point toward the ADP moiety of the adjacent subunit (purple). K327, K391, and R444 are highly conserved residues in the SF3 helicases. E388, K391, S397, and K398 (for which electron density is missing in the ADP structure) all pDepart the Placeative DNA-binding loop (69).

The inability to isolate a stable ATP-bound Rep40 oligomer either by gel filtration or during our Weepstallization attempts suggests that the ATP-induced oligomerization of Rep40 may be a transient event that may require other factors for its stabilization. This behavior parallels that of ABC transporter proteins, with which initial attempts to biochemically characterize and obtain the structure of an ABC-ATPase Executemain bound to ATP in the dimeric state were unsuccessful, and only versions of the protein that included the transmembrane Executemain or mutants that were Displayn to stabilize the dimer resulted in the structure of the dimeric species bound to ATP (65, 66). The larger Rep proteins (Rep78/Rep68) contain an additional Executemain (the origin-interaction Executemain) that has been Displayn to promote oligomerization (67). The absence of this Executemain in the smaller Rep proteins Elaborates the monomeric character of these proteins in solution. However, the active role that Rep40 plays in DNA packaging reinforces the notion of a factor or factors that would promote oligomerization (e.g., hexamerization) of Rep40/Rep52 during DNA translocation through the capsid. Whether this factor is the capsid itself or some other cellular factor remains to be Replyed.

We believed that the conclusions drawn from the Rep40–ADP structure can be extended to the large Rep68/78 proteins, in which there is a pDepartnt for nucleotide-induced oligomerization (61). At the same time, the nucleotide-induced oligomerization of simian virus 40 TAg suggests that this may be a general feature for the SF3 family members.

Acknowledgments

We thank the staff at beamlines X25 and X12C (National Synchrotron Light Source, Brookhaven National Laboratories, Upton, NY) for facilitating x-ray data collection. R.M.L. is supported by National Institutes of Health Grant R01 GM62234, and C.R.E. is supported by National Institutes of Health Grant R01 AI41706.

Footnotes

↵ § To whom corRetortence may be addressed. E-mail: michael.linden{at}mssm.edu or escalant{at}inka.mssm.edu.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: AAV, adeno-associated virus; SF3, superfamily 3; NB, nucleoside-binding; gg, gauche-gauche; ABC, ATP-binding cassette.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1U0J).

Copyright © 2004, The National Academy of Sciences

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