ABSTRACT
Monoclonal antibody (mAb) A20 is the best characterized AAV2 neutralizing mAb [18, 116, 165]. Large quantities of the mAb A20 were obtained from hybridomas cell culture, and A20 Fab fragments were produced and purified by standard methods [289]. Following optimization of the AAV2-A20 Fab ratio, low resolution preliminary images of AAV2-A20 Fab have been obtained. Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of AAV2 with A20. The structure of the AAV2-A20 Fab complex was determined to 28Å resolution using cryo-electron microscopy and image analysis. The known structure of AAV2 [31] and Fab 1CL7 [168] were fitted to the cryo-electron microscope density map. Preliminary results are plausible. Part of the identified A20 footprint is well matched with peptide scanning results (532-541) [116] and mutations at 548, 708 [171]. It is known that A20 is neutralizing, post-attachment, and therefore unlikely to be bound at the primary receptor site. The reconstruction shows mAb A20 binding to the surface off the shoulder of the threefold spike along the twofold axes side, and this region is away from the putative primary receptor binding sites. The reconstruction continues to be improved through addition of EM images, and through improved processing. The crystal structure of heparin-derived hexasaccharides complexed with AAV2 was determined at a resolution of 5.5Å. Here we identify the specific amino acids on the surface of the capsid that facilitate binding to the cell surface receptor heparin sulfate proteoglycan (HSPG). Our data indicate that residues R585 and R588, are primarily responsible for HSPG binding, and therefore for infectivity. They are the minimal necessary and sufficient requirements for HSPG binding. The heparin hexasaccharide also interacts with an additional binding site formed by K-507. No significant conformational change in AAV2 occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the AAV2 signal transduction pathway. HS might function as an accessory molecule, enhancing the efficiency of a second, internalization receptor.
