Demonstration by pulsed neutron scattering that the arrangement of the Fab and Fc fragments in the overall structures of bovine IgG1 and IgG2 in solution is similar

Abstract

The bovine IgG1 and IgG2 isotypes exhibit large differences in effector functions. To examine the structural basis for this, the 12-domain structures of IgG1 and IgG2 were investigated by pulsed neutron scattering using a recently developed camera LOQ. This method reports on the average relative disposition in solution of the Fab and Fc fragments in IgG. The radii of gyration (RG) were found to be similar at 5.64 and 5.71 nm for IgG1 and IgG2 respectively in 100% 2H2O buffers. The two cross-sectional radii of gyration (RXS) were also similar at 2.38-2.41 and 0.98-1.02 nm. Similar values were obtained for porcine IgG. Both bovine IgG1 and IgG2 possess similar overall solution structures, despite sequence differences at the hinge region at the centre of their structures. An automated computer survey of possible IgG structures was developed, in which coordinates for the two Fab fragments were displaced in a two-dimensional plane relative to those of the Fc fragment in 0.25 nm steps. The scattering curves calculated from these structures were found to be sensitive to relative displacements of the three fragments, but not on their rotational orientation about their longest axes. Good agreement with the solution scattering data was obtained with a planar IgG model in which the C-terminus of the CH1 domain of Fab was 3.6 nm from the N-terminus of Fc in both IgG1 and IgG2, with a precision of 0.7 nm. Energy refinement showed that this spatial separation is compatible with the hinge sequences of bovine IgG1 and IgG2. The results show that multidomain protein structures can be modelled using LOQ data, and that a long hinge sequence does not necessarily reflect a large distance between Fab and Fc. The steric accessibility of Fc sites for interactions with cell-surface Fc receptors and C1q of complement is shown to be generally similar for IgG1 and IgG2, and the difference in effector function between IgG1 and IgG2 is probably based on deletions in the IgG2 hinge sequence.

Measurement of the localised plasmon penetration depth for gold nanoparticles using a non-invasive bio-stacking method

“…The derived step length range of 17.5 nm is consistent with Guinier analysis of IgG by Mayans et al 18 ;…”

The role of polymer nanolayer architecture on the separation performance of anion‐exchange membrane adsorbers: I. Protein separations

“…Serum albumin is reported to be an oblate ellipsoid with dimensions of 14 nm  4 nm (Bendedouch and Chen, 1983). IgG is reported to have a cross-sectional diameter of 4.8 AE 0.3 nm (Mayans et al, 1995). In both cases, one dimension of the protein is similar in size to the estimated interchain distances reported in Table II.…”

Factor VIIa and the extracellular domains of human tissue factor form a compact complex: A study by X‐ray and neutron solution scattering

“…Ultimately the combination of the scattering curves in Figs. 1-3 with a recently-developed method of automated curve fitting of domain models to the scattering curves (which is constrained using known atomic structures for individual domains [22,24]) should permit the quantitative testing of sterically-allowed domain structures in FVIIa, sTF and their complex to produce improved low-resolution structures compared to those presented in Fig. 4.…”

“…Use of the average of the X-ray and neutron values will approximately represent a structure at infinite contrast by analogy with the Stuhrmann relationship, in which this contrast effect is minimised [9,10,25]. As the neutron intensities had been standardized using a deuterated polymer standard [23], for recombinant human IgE-Fc (Mr of 75,300) [22] and 0.181 for bovine IgG1 and IgG2 (Mr of 144,000) [24]. It is concluded that the complex was formed as expected.…”