Three-dimensional structure of an intact human immunoglobulin.

Abstract

We have examined the low-resolution structure of a complete human IgGi using known domain coordinates from crystallographic investigations of immunoglobulin fragment structures. Our results indicate that the Fc ortion of this molecule has a structure similar to that of an isolated Fc fragment, with the carbohydrate moiety playing a central role as the principal contact between the CH2 domains. Carbohydrate also forms a large part of the interface between the Fc and Fab regions. The relative orientations of the variable and constant portions of the Fab regions are intermediate between those reported previously, emphasizing the flexibility of the switch region. These data do not support a two-state allosteric model such as has been proposed for antibody effector functions. Most of the information available at present on the threedimensional structure of immunoglobulins (Igs) has come from studies of isolated fragments (1-3). Progress in the determination of structure of intact Igs has been generally disappointing. The protein Dob, a human IgGl(K) cryoglobulin, was the first crystalline intact Ig to be analyzed by x-ray diffraction (4). The crystals are sensitive to x-irradiation and show evidence of disorder in the diffraction pattern, with few useful reflections beyond 6 A. An electron density map at this resolution showed the overall molecular boundary, on the basis of which a Tshaped model was proposed (5). This model was independently supported by an analysis of electron micrographs of sections of the Dob crystals (6, 7).The human myeloma protein Mcg [IgGl(X)] has also been crystallized, and the crystals diffract to a resolution of 3.5 A (8), but there has been no reported solution of its three-dimensional structure.A third human myeloma protein (IgGi), Kol, has been crystallized and shown to diffract to spacings of 3.5 A (9). An electron density map at 5-A resolution has been reported in which the two Fab arms are clearly visible with an angle of 1200 between them (10). Unfortunately, the Fc part of the molecule is not at all visible in this map, probably because of disorder in the crystal.Progress in the determination of structure of Ig fragments has been more rapid. the structures of two Fabs (11,12), an Fc (13,14), and a number of Bence-Jones proteins (15-17) have been reported. As a result, we now know separately the structures of all the component domains of an IgG molecule. When corresponding domains from different Igs have been compared, they have been found to be similar (refs. 10, 17, and 18; unpublished*). These observations, together with the results of amino acid sequence analyses, strongly suggest that, even in the variable parts of the molecule, the three-dimensional structures of the domains will be highly conserved.The quaternary relationships between domains are not, however, invariant. Substantial differences have been observed in the relative orientation of the variable and constant regions (elbow bend) of the Fabs (ref. 10; unpublished*). Furthermore, solution studies have demonst...

Detection and Quantification of Protein Biomarkers from Fewer than 10 Cells

“…A key feature of ultramicroarrays is that the molecular population of antibodies is on the order of several hundred thousand molecules in a 5-mdiameter spot. This was calculated based on the dimension of an IgG to be 14.5 ϫ 8.5 ϫ 4 nm (29,30) and an optimal packing of IgG molecules. Therefore a 5-m spot will create the opportunity for at least 3-4 logs of dynamic range while retaining the advantages of ultraminiaturization.…”

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry

“…Its crystalline structure has approximate dimensions of 142 Å Â 85 Å Â 38 Å [2]. Because of the delicate location of the antigen-binding site located in the two variable domains in each Fab fragment, the interfacial orientation of the antibody on the support interface hugely affects the site accessibility.…”

Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1

“…While well over 200 structures of antibody fragments, mainly Fab and Fab H , have been determined, crystals of intact antibodies have only been reported eight times: Dob (Terry et al, 1968), Mcg (Edmundson et al, 1970), Kol (Palm & Colman, 1974), Zie (Ely et al, 1978), Mab231 (Larson et al, 1991;Harris et al, 1992), Mab4B7 (Stura et al, 1994), Mab24-404.1 and Mab61.1.3 (Harris et al, 1995 (Klein et al, 1981). The crystal structure determinations of Dob and Mcg revealed compact symmetrical planar T shapes (Silverton et al, 1977;Rajan et al, 1983;Guddat et al, 1993).…”