Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors

Abstract

Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C gamma 3* and the IgG1 mutant gamma/C gamma 3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of the Ig in the Fab segments. Eosin-DNS bound in these Fab sites served as the acceptor probe in these studies. Both Ig have a carboxy (C)-terminal domain (C gamma 3*) which contains genetically introduced cysteine residues. Modification of these cysteine sulfhydryls with fluorescein maleimide provided donor probes near the C-terminal ends of the Ig in the Fc segment. Energy transfer between the C-terminal and N-terminal ends was compared for these two Ig in solution and when they were found to their respective high-affinity receptors on plasma membranes: IgE-Fc epsilon RI on RBL cell membranes and IgG1-Fc gamma RI on U937 cell membranes. Previous energy-transfer measurements with these probes yielded an average end-to-end distance of 71 A for IgE in solution and 69 A for IgE bound to Fc epsilon RI, indicating that in both situations IgE is bent such that the axes of the Fab segments and the axis of the Fc segment do not form a planar Y-shape [Zheng, Shopes, Holowka, & Baird (1991) Biochemistry 30, 9125]. In the current study we found the average end-to-end distance for IgG1 in solution is 75 A and greater than or equal to 85 A for IgG1 bound to Fc gamma RI, suggesting an average bend conformation for IgG1 as well. The contributions of segmental flexibility to the average distances were assessed directly by measuring the efficiency of energy transfer as a function of variations in donor quantum yield caused by a collisional quencher and using these data to extract a Gaussian distribution of end-to-end distances. The distribution average (rho) and half-width (hw) were determined to be as follows: rho = 75 A, hw = 24 A for IgE in solution; rho = 71 A, hw = 12 A for IgE bound to Fc epsilon RI; and rho = 100 A, hw = 88 A for IgG in solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain.

“…We note first that the derived composition of the complex does not depend on the accuracy with which the partial specific volumes are known-that is to say, on the carbohydrate content of the constituents-because the buoyant molecular weight, determined from the sedimentation equilibrium distribution, is the sum of the buoyant molecular weights of the constituent glycoproteins. The It has been suggested that IgE may be bent (26,27), and this distortion could occur, at least in part, within the CE2-CE3 linker. The consequences of this were investigated by using the extended model.…”

“…and S.J.P., unpublished results). In the absence of more detailed structural information, this distortion is generally represented as being evenly distributed between all the linker regions separating the immunoglobulin domains (1,26,27 The hydrodynamic evidence limits the range of possible models for the complex of IgE-Fc and sFcsRIa to those in which the domains of the IgE-Fc overlap with those of the sFceRla (Fig. 4 g-j), although two T-shaped models (Fig.…”

A Conformational Rearrangement upon Binding of IgE to Its High Affinity Receptor

“…Extensive energy transfer studies suggest that IgE is bent at the junction of its C⑀2 and C⑀3 domains (19 -23). Furthermore, the bent structure of IgE in solution appears to be similar in conformation to receptor-bound IgE (19,23). These data suggest that the binding site for Fc⑀RI could be on the convex surface of IgE and thus the concave surface would not be available for the binding of a second receptor.…”

“…However, no direct evidence has been presented to confirm a conformational rearrangement upon binding. Furthermore, later energy transfer studies suggest that the bent conformation of IgE does not change significantly upon binding (19,23).…”

Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice

“…This means that these small molecules have to present at least two epitopes for cross-linking the IgE, which could be difficult from a stereochemical point of view. Moreover, through the absence of a hinge region, IgE is rather rigid, and its flexibility is limited when bound to Fc⑀RI, where IgE is fixed in a bent shape (7,8). The cross-linking situation is even more stringent in the case of B lymphocyte activation, because each B cell expresses only one type of Ig with one epitope specificity.…”