Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography

Abstract

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 Å resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few angstroms by fitting atomic models of CPMV 4 and Fab (Kol) 5 to reconstructed density maps.Comoviruses are plant viruses whose structural and biological properties are strikingly similar to the animal picornaviruses 6,7 . For example, the protein capsids of CPMV, the type member of the comovirus group, and poliovirus, the most extensively studied picornavirus, are comparable (Fig. 1a). The CPMV coat contains 60 copies each of large (relative molecular mass 42,000 (M r 42K) and small (24K) protein subunits arranged with icosahedral symmetry. Each large subunit (L) is composed of two antiparallel β-barrel domains which are positioned close to the icosahedral 3-fold axes. The β-barrel domain at the amino-terminal end of L corresponds to the VP2 subunit in poliovirus and the β-barrel at the carboxy-terminal end of L corresponds to VP3 in poliovirus. The small subunits (S), packed near the icosahedral 5-fold axes, correspond to VP1 in poliovirus.CPMV is strongly immunogenic when injected into rabbits or mice. We prepared nine monoclonal antibodies against CPMV using standard methods 8 . The binding properties of these antibodies were analysed by enzyme-linked immunosorbent assay (ELISA) and all nine would only bind to virus particles presented in a double antibody sandwich-type ELISA © 1992 Nature Publishing Group Correspondence to: John E. Johnson. G.W. and C.P. are joint first authors. in which the virus is bound to a polyclonal antibody attached to the plastic plate. Denaturation of the virus by its direct adsorption onto a plastic surface destroys the antigenic sites for all nine monoclonal antibodies. Only monoclonal antibodies 5B2 and 10B7 reacted strongly with CPMV that was free in solution (antibody binding was assayed by electron microscopy analysis of negatively stained complexes marked with gold-labelled anti-mouse immunoglobulin). Fab fragments from monoclonal antibody 5B2 were prepared by digestion of IgGs with insoluble...

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment

“…For the CPV: Fab structure, atomic level information is available for the structure of CPV as well as of an Fab model with an elbow angle similar to that observed in the image reconstruction of the complex. Thus, the accuracy with which the final structure was known, after refinement of the rigid body components [10,16], permitted placement of individual amino acid groups with confidence, although the resolution of the EM reconstruction was only 23Å (Fig. 4).…”

Characterization of a Conformational Epitope on Hepatitis B Virus Core Antigen and Quasiequivalent Variations in Antibody Binding

“…The first cryo-EM investigations of capsids decorated with antibodies were performed over ten years ago (27, 38a, 38). When feasible, molecular modeling enhances the interpretability of the resulting density maps (38). This area of research, recently reviewed by Smith (31), now covers numerous applications, many of which have addressed mechanisms of antibody-based neutralization (e.g., see references 19, 26, and 41), while others have identified the locations of specific proteins (27) or individual peptides (14,33,34).…”

“…However, this approach imposes daunting requirements in terms of the amount of material, crystallinity, and data analysis. Here we demonstrate an alternative approach based on cryo-electron microscopy (cryo-EM) of Fab-decorated antigens (31,38), which requires less material by Ͼ3 orders of magnitude and has no need for crystals. The basic idea is as follows: provided that the antigen structure is known to high resolution, a cryo-EM structure of the antigen-Fab complex at moderate resolution, probed by molecular modeling with a generic Fab structure from the protein database, contains sufficient information to allow identification of the peptides that make up the epitope.…”

Structure of a Human Rhinovirus Complexed with its Receptor Molecule

“…tron microscopy in a manner similar to that reported here (45); however, neither the structure of the virus nor the antibody was known in atomic detail. Recently, the structure of a complex ofcowpea mosaic virus and a bound monoclonal antibody Fab fragment was determined with EM (46). In that case, an atomic resolution structure of cowpea mosaic virus was known, permitting determination of the antibody footprint on the viral surface.…”

“…The parts of the predicted ICAM-1 structure that contact HRV-14 are the amino-terminal four residues and loops B-C (residues 24-26), D-E (residues [45][46][47][48][49], and F-G (residues 71 and 72; see Fig. 2 Enlarged view of the residues in the ICAM-1 footprint showing the residues (hatched areas) that, when mutated, affect viral attachment (Right) (9), and the residues (stippled areas) altered in structure by the binding of antiviral compounds that inhibit attachment and uncoating (Left) (40).…”