The three-dimensional structure of an intact monoclonal antibody for canine lymphoma

Abstract

Crystal structures of Fab antibody fragments determined by X-ray diffraction characteristically feature four-domain, beta-barrel arrangements. A human antibody Fc fragment has also been found to have four beta-barrel domains. The structures of a few intact antibodies have been solved: in two myeloma proteins, the flexible hinge regions that connect the Fc to the Fab segments were deleted so the molecules were non-functional, structurally restrained, T-shaped antibodies; a third antibody, Kol, had no hinge residues missing but the Fc region was sufficiently disordered that it was not possible to relate its disposition accurately with respect to the Fab components. Here we report the structure at 3.5 A resolution of an IgG2a antitumour monoclonal antibody which contains an intact hinge region and was solved in a triclinic crystal by molecular replacement using known Fc and Fab fragments. The antibody is asymmetric, reflecting its dynamic character. There are two local, apparently independent, dyads in the molecule. One relates the heavy chains in the Fc, the other relates the constant domains of the Fabs. The variable domains are not related by this 2-fold axis because of the different Fab elbow angles of 159 degrees and 143 degrees. The Fc has assumed an asymmetric, oblique orientation with respect to loosely tethered yet almost collinear Fabs. Our study enables the two antigen-binding segments as well as the Fc portion of a functional molecule to be visualized and illustrates the flexibility of these immune response proteins.

Scanning tunneling microscopy of monoclonal immunoglobulin G

“…The overall shape of the molecule is conserved and the lateral sizes agree with the expected size of an IgG. The image is even closer to the present models [24] and to the crystal structure of intact IgG [21] and the measured vertical size is close to 16 À.…”

“…In several occasions, however, we imaged objects like the one shown in figure 3, panel A, clearly composed of two similar structures (80 x 40 À) resembling the Fab fragments of an IgG associated with a lower part whose sides measure 60 À interpreted as the Fc fragment. The slight asymmetry of the image compares with that found in the recently published crystallographic study of an IgG with a normal Hinge region [21]. The apparent height of the structure shown in the figure is too small for an IgG (less than 10 À).…”

Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

“…The density related to the variable domains of the Fab fragments was almost as strong as the density of the glycoprotein shell, indicating ∼90% occupancy of the 120 binding sites (87% for X1, 93% for X2). The density related to the constant regions of Fab CR4354 is only about 0.7-fold as strong as the variable region, suggesting flexibility of the elbow angle between variable and constant domains, as often observed with antibody structures (23)(24)(25)(26)(27).…”

West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody

“…Indeed, a conformational change in the elbow angle of Ϸ40°relative to that in the crystal structure of the Fab E16ϩDIII complex (Fig. 4) shows flexibility, as is often found in antibody structures (30)(31)(32)(33). Presumably, the very similar elbow angles in the Fab molecules bound to the independent binding sites DIII-B and DIII-C in the cryo-EM structure represent the lowest energy conformation, whereas the x-ray structure may have a slightly higher energy to achieve better crystal packing.…”