Adeno-Associated Virus Receptor-Binding: Flexible Domains and Alternative Conformations through Cryo-Electron Tomography of Adeno-Associated Virus 2 (AAV2) and AAV5 Complexes

Figures

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  FIG 1. Cryo-ET of AAV-receptor complexes. (A) Slice through a tomogram of AAV2/PKD1-2. AAVR densities are indicated by the red arrows. (B) Slice through a tomogram of AAV5/PKD1-2. (C) Fourier shell correlation (FSC) curves for the subvolume average reconstructions from cryo-ET with estimated resolutions of 20 Å for AAV2/PKD1-2 (blue) and 25 Å for AAV5/PKD1-2 (green). Scale bars: 1,000 Å.

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  FIG 2. Classification of AAV2/PKD1-2 trimer spikes. (A) Orientating panel showing a surface representation of AAV2 from the atomic structure, PDB ID 1LP3. Scale bar: 100 Å. The circle shows the area that was classified by subvolume averaging, and the axes of symmetry are labeled as 2, 3, and 5. (B) Global tomographic subvolume average of AAV2/PKD1-2 trimeric spike as solid surface (1) and translucently (2) overlaid on the atomic model of a subunit trimer from the prior single-particle reconstruction (PDB ID 6nz0). The viral proteins are shown in gray ribbons, and two of the PKD2 domains are colored blue, while one is rainbow colored by residue number from N (blue) to C (red). (C) Classification of the region outside the PKD2 domain revealed 4 distinct conformations for PKD1. (D) The 4 PKD1 domain conformations were segmented and overlaid, C and PKD1 atomic models (shades of orange) were fit to the 4 classes seen in panel C.

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  FIG 3. Classification of AAV5/PKD1-2 trimer spikes, oriented as in Fig. 2A. (A) Global tomographic subvolume average of the AAV5/PKD1-2 trimeric spike, alone (1) and overlaid (2) with the atomic structure from single-particle reconstruction (PDB ID 7kp3) in which the PKD1 domain of AAVR was seen. The viral proteins are shown in gray ribbons, and two of the PKD1 domains are colored orange, while one is rainbow colored by residue number from N (blue) to C (red). Scale bar: 100 Å. (B) Classification of the region outside the PKD1 domain revealed 3 distinct conformations for PKD2. (C) The 3 PKD2 domain conformations were segmented and are shown overlapping (shades of blue).

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  FIG 4. Conformational variability of AAV2/PDK1-2 and AAV5/PKD1-2 structures. The virus surfaces are viewed along a 2-fold axis (top) and tangentially (bottom), with 3-folds and 5-folds marked as triangles and pentagons, respectively. In each, the AAVR receptor is shown bound to one of the symmetry-equivalent regions on the virus surface. (A) AAV2, with the AAVR PKD2 domain (blue) tightly bound and the preceding PDK1 domain in several orientations (shades of orange) as determined from the subvolume tomography. (B) In its complex with AAV5, a tightly bound conformation of AAVR PKD1 (orange) is followed by PDK2 in three orientations (shades of blue) that are more constrained than PKD1 in panel A.

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  FIG 5. Unmodeled density inside the AAV2 capsid. (A) The inner surface of a subvolume average of the AAV2/PKD1-2 complex surrounding a 3-fold, viewed outwards. The circled region is highlighted in panels C to E. (B) The corresponding subvolume of the AAV5/PKD1-2 complex. (C) The 2.8-Å single-particle reconstruction (EMD9672) and model (PDB ID 6ihb) of an AAV2-AAVR (PKD1-5) complex (58). The map clearly shows βA proceeding left-to-right to the hairpin turn at residue 237 as has been seen in AAV2 crystal structures (1). (D) In the 2.4-Å single-particle reconstruction of an AAV2-AAVR complex (PKD1-2; EMD0553) (44), this configuration of βA is not apparent, as illustrated by overlaying the (mismatched) model PDB ID 6ihb (24, 58). (E) Overlaid on panel D is the newly observed AAV2/PKD1-2 subvolume average tomographic density, suggesting that the unmodeled density extending inward from the surface is a partially ordered configuration of upstream residues. It would be an alternate to the βA strand, connecting to βB at residue 237.

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  FIG 6. Single-particle analysis of AAV5/PKD1-2. (A) Representative micrograph of the single-particle AAV5/PKD1-2 data. Scale bar: 1,000 Å. (B) Protomer of the AAV5 PKD1-2 complex extracted from the SPA reconstruction colored by local resolution as estimated by ResMap. Scale bar: 25 Å. (C) Fourier shell correlation (FSC) curves for the AAV5/PKD1-2 SPA reconstruction. Curves are shown for the unmasked half maps (blue) and masked (green) with an estimated resolution of 2.8 Å.

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  FIG 7. PKD1 structure from SPA of AAV5 complexes. (A) Comparison of the Silveria et al. (60) and Zhang et al. (58) models of AAVR PKD1 as bound to AAV5. Arrows indicate the discrepancies between the N-terminal residues of the two models. (B) Models in panel A compared to the cryo-EM SPA map determined here. The dashed line indicates unmodelled features at low contour. (C) Models in panel A compared to the Zhang et al. map, the bracket indicating part of the dashed region modeled by Zhang et al., but not Silveria et al. (D) At high contour, the current reconstruction supports the Silveria model as at least a dominant conformer. The first strand is labeled βA. (E) Low contouring of the new map shows two putative β strands (red) continuing the β sheets on the top and bottom of the PKD1 structure. In this model βA is in the same position as in (D). This panel shows The most probable interpretation, but panels F and G present an alternative. (F) Homologs in the PDB have structures similar to both the Silveria and Zhang models and, less frequently, to PDB ID 6aem. Overlay of this structure at 1.3-Å resolution shows how the longer of the unmodeled regions could be an alternative conformer of…