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Abstract
The structure of an intact anti-tumor, murine monoclonal antibody, Mab231 (IgG2a$\kappa$), has been determined by molecular replacement and refined in a triclinic unit cell to a resolution of 2.8 A. All segments of the antibody are visible in electron density maps, although portions of the hinge peptides remain ambiguous. The two oligosaccharide components of the molecule were discerned and refined as well. Mab231 was raised against, and binds specifically to canine lymphoma cells.
The antibody structure is asymmetric, illustrating the flexibility of these immune response proteins. The Fc has assumed an oblique orientation with respect to loosely tethered, yet almost collinear Fabs. There are, however, two local, apparently independent, near exact dyad axes in the molecule. One relates the heavy chains in the Fc, the other relates the constant domain pairs of the Fabs. The variable domains exhibit no symmetry relationship, but only as a consequence of differing Fab elbow angles of 159$\sp\circ$ and 143$\sp\circ$. The CDRs of the two antigen binding segments are virtually superimposable, with only minor differences due to limited side chain movement. The C$\sb{\rm H}$2 domains of the Fc segment exhibit substantial rigid body rotations with respect to the human Fc starting model, demonstrating flexibity at the switch peptides that join C$\sb{\rm H}$2 to C$\sb{\rm H}$3. The crystallographic study, described here, enables the two Fab segments as well as the Fc portion of an entire functional immunoglobulin to be visualized. The unique structure seen in these crystals is presumably a product of the inherent segmental flexibility of the antibody and the lattice interactions that stabilize this particular distribution of domains. The structure of the antibody should likely be regarded as a "snapshot" of only one of many possible conformations occurring in solution.
Using a PEG 3350 screen combined with detergents, and developed from experiments with the IgG2a$\kappa$ anti-canine lymphoma antibody, crystals were obtained of two additional immunoglobulins, an anti-phenytoin and an anti-phenobarbital antibody. A complex between the phenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG1$\kappa$ antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic and structural analyses are in progress.
