Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Valentina A Spiteri,Margaret Goodall,James Doutch,Robert P Rambo,Jayesh Gor,Stephen J Perkins

doi:10.1016/j.jbc.2021.100995

Abstract

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s020,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier RG values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.

Highlights

Immunoglobulin immunoglobulin G subclass 3 (IgG3) is one of the four subclasses IgG1 to IgG4 that make up the human IgG antibodies
The first experimentally determined molecular structure for a full-length IgG3 antibody was determined by this joint analytical ultracentrifugation (AUC), small-angle Xray scattering (SAXS), and small-angle neutron scattering (SANS) study of human myeloma IgG3, combined with an atomistic scattering curve fit method based on molecular dynamics and Monte Carlo simulations [27]
Its solution structure was shown to possess a semirigid hinge with little disorder that resulted in a wide separation between the Fab and Fc regions in IgG3

Summary

Introduction

Immunoglobulin IgG3 is one of the four subclasses IgG1 to IgG4 that make up the human IgG antibodies. In the resulting size distribution analyses c(s), a clear monomer peak that monitored the overall IgG3 solution structure was observed at average s20,w values of 5.82 ± 0.06 S for glycosylated IgG3 and 6.29 ± 0.05 S for deglycosylated IgG3 in light water (Fig. 4A; Table 1).

Results

Conclusion