Adeno-associated virus receptor complexes and implications for adeno-associated virus immune neutralization

Figures

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  Figure 1. Comparison of glycan attachment sites overlaid on the structure of AAV2, from Stagg et al. (2022). Equivalent sites are shown surrounding one of the three-fold axes of symmetry of AAV. Arrows point to space-filling representations of fondaparinux, a heparin analog from the AAV2 cryo-EM complex (Xie et al., 2017), sialic acid (SIA, magenta carbons) from the AAV1 crystal structure (Huang et al., 2016), and galactose (gal; green carbons) from the cryo-EM AAV9 structure (Penzes et al., 2021), the latter two overlapping. Two arginines (585 & 588), important in AAV2 heparan-binding, are colored blue (Kern et al., 2003). Additional glycan attachment interactions for AAV1, AAV3B, and AAV5 have also been inferred through crystallography or mutational studies (Lerch and Chapman, 2012; Afione et al., 2015; Huang et al., 2016). Structures were overlaid by superimposing the AAV VP3 capsid proteins using the SSM sequence-structure alignment as implemented in Pymol (DeLano, 2002; Krissinel and Henrick, 2004; The PyMOL Molecular Graphics System, 2.0, 2015), and then expanding the icosahedral symmetry.

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  Figure 2. AAV receptor binding sites. Most AAV serotypes bind primarily to the PKD2 domain (blue) of AAVR as seen in the 2.4 Å cryo-EM structure of the complex (Meyer et al., 2019). A smaller clade binds to the PKD1 domain (green) as exemplified in the 2.5 Å cryo-EM structure (Silveria et al., 2020). In the right panels, the AAVR domains have been removed to reveal the contact footprints (4.5 Å cut-off) for all of the binding surfaces that are related by the viral symmetry axes (numbered arrows). Prominent features of AAV’s surface are annotated.

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  Figure 3. Cryo-ET reveals variable locations for neighboring PKD domains that are not strongly bound to AAV. (A) The region shown in panels (B,C) is outlined on part of the surface of the AAV2 crystal structure (PDB ID 1LP3) (Xie et al., 2002). Spikes are seen surrounding a 3-fold axis, with neighboring 2- and 5-fold axes shown for reference. (B) Cryo-ET subtomograms for an AAV2-PKD12 complex have been averaged within four classes that are superimposed with different colors (yellow through red) for the PKD1 domain that is in variable orientation. Regions in common between the classes are colored grey, and comparison with panel (A) shows the added mass of PKD2 running up from near the 2-fold to above the 3-fold spike. (C) Cryo-ET subtomogram class averages for AAV5-PKD12 that have been similarly represented to show the variable orientations of PKD2 (shades of blue) emanating from a symmetry-related 2-fold axis behind the spike. PKD1 is bound in a single configuration, adding to the grey volume (cf. panel A) between the 5- and 2-fold axes. Panels are taken with permission from Hu et al. (2022).

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  Figure 4. Conflict between neutralizing mAb A20 and the AAVR receptor from Stagg et al. (2022). Backbone traces of AAVR PKD2 domain (violet) and the A20 Fab’ structure (salmon) from the structures of AAV2 complexes are overlaid on the surface of the virus (McCraw et al., 2012; Xie et al., 2017; Meyer et al., 2019). Conflict, highlighted in the inset, occurs between the N-terminal residues of PKD2 and the CDR loops of mAb A20 above the spur extending from AAV2’s 3-fold spike. PKD1 was not seen in the single-particle cryo-EM structures (Meyer et al., 2019; Zhang et al., 2019), but was seen at low resolution in recent cryo-electron tomography (Cryo-ET) (Hu et al., 2022). PKD1 is in four orientations extending beyond PKD2 Asn405, adding extensively to the overlap with mAb A20. A20 is the only antibody whose complex with AAV is deposited in the structural databases. For structures to which we do not have access (Figure 5), one may still highlight AAV residues reported to be in contact. (Those within 4 Å of A20 are rainbow colored here). Then one could judge how each antibody would be placed to make the identified contacts, and whether there would likely be receptor-overlap.

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  Figure 5. Epitopes of additional anti-AAV monoclonal antibodies. This figure is similar to that in Stagg et al. (2022), but updated to include the most recent structures of AAV-receptor complexes (violet backbone traces), with PKD1 resolved for AAV5 and PKD2 resolved for AAVR complexes with other serotypes (Meyer et al., 2019; Zhang et al., 2019; Silveria et al., 2020; Xu et al., 2022). Surface residues of several AAV serotypes are rainbow-colored according to residue number if reported to be in contact with the mAb indicated in the panel label, as inferred from cryo-EM structures at the noted resolutions (Xie et al., 2002, 2011; Nam et al., 2007; DiMattia et al., 2012; Gurda et al., 2012, 2013; Tseng et al., 2015; Bennett et al., 2018; Giles et al., 2018; Jose et al., 2019; Zhang et al., 2019; Silveria et al., 2020). Other parts of the surface are cream colored, except as noted below. Wheat-colored regions (panels F,G) were reported as occluded by antibody-binding (Bennett et al., 2018; Giles et al., 2018). Known glycan attachment sites are indicated as overlaid spheres for sialic acid or stick-model for fondaparinux (panels A–I) (Huang et al., 2016; Xie et al., 2017), while amino acids…