Structure of adeno-associated virus type 2 Rep40-ADP complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases

Abstract

We have determined the structure of adeno-associated virus type 2 (AAV2) Rep40 to 2.1-A resolution with ADP bound at the active site. The complex crystallizes as a monomer with one ADP molecule positioned in an unexpectedly open binding site. The nucleotide-binding pocket consists of the P-loop residues interacting with the phosphates and a loop (nucleoside-binding loop) that emanates from the last strand of the central beta-sheet and interacts with the sugar and base. As a result of the open 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 explains the strong tendency by members of this family such as simian virus 40 TAg to oligomerize after binding ATP.

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 open 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 consecutive residues, 402–407, missing. There are two secondary elements: a βe strand that is part of a three-stranded β-sheet together with strands βc–βd and a 3₁₀-helix h2 that is between β2 and β3. (b) A 2F_o – F_c simulated annealing omit map showing the electron density for the bound ADP at 1.5 σ.

Fig. 2.

Rep40–ADP interactions. (a) The nucleotide binding pocket in Rep40. (b) Stereoview of the active site. The dotted 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 regions 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 shown as a surface representation (salmon).

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 differences are observed in response to ADP binding. (b) Plot of the average difference distance (Å) versus residue number for the two superimposed molecules. Four regions of differences 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. Differences in the β-hairpin 1 (βa–βb) loop may be caused by the lack of crystal contacts in the ADP-bound form of the protein.

Fig. 4.

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

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 precede the putative DNA-binding loop (69).