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Human immunoglobulin G (IgG) exists as four subclasses IgG1-4, each of which has two Fab subunits joined by two hinges to a Fc subunit. IgG4 has the shortest hinge with 12 residues. The Fc subunit has two glycan chains, but the importance of glycosylation is not fully understood in IgG4. Here, to evaluate the stability and structure of non-glycosylated IgG4, we performed a multidisciplinary structural study of glycosylated and deglycosylated human IgG4 A33 for comparison with our similar study of human IgG1 A33. After deglycosylation, IgG4 was found to be monomeric by analytical ultracentrifugation; its sedimentation coefficient of 6.52 S was reduced by 0.27 S in reflection of its lower mass. X-ray and neutron solution scattering showed that the overall Guinier radius of gyration RG and its cross-sectional values after deglycosylation were almost unchanged. In the P(r) distance distribution curves, the two M1 and M2 peaks that monitor the two most common distances within IgG4 were unchanged following deglycosylation. Further insight from Monte Carlo simulations for glycosylated and deglycosylated IgG4 came from 111,382 and 117,135 possible structures respectively. Their comparison to the X-ray and neutron scattering curves identified several hundred best-fit models for both forms of IgG4. Principal component analyses showed that glycosylated and deglycosylated IgG4 exhibited different conformations from each other. Within the constraint of unchanged RG and M1-M2 values, the glycosylated IgG4 models showed more restricted Fc conformations compared to deglycosylated IgG4, but no other changes. Kratky plots supported this interpretation of greater disorder upon deglycosylation, also observed in IgG1. Overall, these more variable Fc conformations may demonstrate a generalisable impact of deglycosylation on Fc structures, but with no large conformational changes in IgG4 unlike those seen in IgG1., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Spiteri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

FcγRI (CD64) is the only high-affinity Fcγ receptor found on monocytes, macrophages, eosinophils, neutrophils and dendritic cells. It binds immunoglobulin G (IgG) antibody-antigen complexes at its Fc region to trigger key immune responses. CD64 contains three immunoglobulin-fold extracellular domains (D1, D2 and D3) and a membrane-spanning region. Despite the importance of CD64, no solution structure for this is known to date. To investigate this, we used analytical ultracentrifugation, small-angle X-ray scattering, and atomistic modelling. Analytical ultracentrifugation revealed that CD64 was monomeric with a sedimentation coefficient s020,w of 2.53 S, together with some dimer. Small-angle X-ray scattering showed that its radius of gyration RG was 3.3-3.4 nm and increased at higher concentrations to indicate low dimerization. Monte Carlo modelling implemented in the SASSIE-web package generated 279,162 physically-realistic trial CD64 structures. From these, the scattering best-fit models at the lowest measured concentrations that minimised dimers revealed that the D1, D2 and D3 domains were structurally similar to those seen in three CD64 crystal structures, but showed previously unreported flexibility between D1, D2 and D3. Despite the limitations of the scattering data, the superimposition of the CD64 solution structures onto crystal structures of the IgG Fc-CD64 complex showed that the CD64 domains do not sterically clash with the IgG Fc region, i.e. the solution structure of CD64 was sufficiently compact to allow IgG to bind to its high-affinity Fcγ receptor. This improved understanding may result in novel approaches to inhibit CD64 function, and opens the way for the solution study of the full-length CD64-IgG complex., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 s 0 20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R G 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is composed of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG; however, their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. After deglycosylation using peptide:N-glycosidase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s 0 20,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass after glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. Although the radius of gyration (R G ) was unchanged, the cross-sectional radius of gyration (R XS-1 ) increased by 0.1 nm, and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed after deglycosylation. To explain these changes, atomistic scattering modeling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 x-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, whereas the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the Fcγ receptor in deglycosylated IgG1., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Small angle x-ray and neutron scattering are techniques that give solution structures for large macromolecules. The creation of physically realistic atomistic models from known high-resolution structures to determine joint x-ray and neutron scattering best-fit structures offers a, to our knowledge, new method that significantly enhances the utility of scattering. To validate this approach, we determined scattering curves for two human antibody subclasses, immunoglobulin G (IgG) 1 and IgG4, on five different x-ray and neutron instruments to show that these were reproducible, then we modeled these by Monte Carlo simulations. The two antibodies have different hinge lengths that connect their antigen-binding Fab and effector-binding Fc regions. Starting from 231,492 and 190,437 acceptable conformations for IgG1 and IgG4, respectively, joint x-ray and neutron scattering curve fits gave low goodness-of-fit R factors for 28 IgG1 and 2748 IgG4 structures that satisfied the disulphide connectivity in their hinges. These joint best-fit structures showed that the best-fit IgG1 models had a greater separation between the centers of their Fab regions than those for IgG4, in agreement with their hinge lengths of 15 and 12 residues, respectively. The resulting asymmetric IgG1 solution structures resembled its crystal structure. Both symmetric and asymmetric solution structures were determined for IgG4. Docking simulations with our best-fit IgG4 structures showed greater steric clashes with its receptor to explain its weaker FcγRI receptor binding compared to our best-fit IgG1 structures with fewer clashes and stronger receptor binding. Compared to earlier approaches for fitting molecular antibody structures by solution scattering, we conclude that this joint fit approach based on x-ray and neutron scattering data, combined with Monte Carlo simulations, significantly improved our understanding of antibody solution structures. The atomistic nature of the output extended our understanding of known functional differences in Fc receptor binding between IgG1 and IgG4., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Human IgG2 antibody displays distinct therapeutically-useful properties compared with the IgG1, IgG3, and IgG4 antibody subclasses. IgG2 is the second most abundant IgG subclass, being able to bind human FcγRII/FcγRIII but not to FcγRI or complement C1q. Structural information on IgG2 is limited by the absence of a full-length crystal structure for this. To this end, we determined the solution structure of human myeloma IgG2 by atomistic X-ray and neutron-scattering modeling. Analytical ultracentrifugation disclosed that IgG2 is monomeric with a sedimentation coefficient ( s 20, w 0 ) of 7.2 S. IgG2 dimer formation was ≤5% and independent of the buffer conditions. Small-angle X-ray scattering in a range of NaCl concentrations and in light and heavy water revealed that the X-ray radius of gyration ( R g ) is 5.2-5.4 nm, after allowing for radiation damage at higher concentrations, and that the neutron R g value of 5.0 nm remained unchanged in all conditions. The X-ray and neutron distance distribution curves ( P ( r )) revealed two peaks, M1 and M2 , that were unchanged in different buffers. The creation of >123,000 physically-realistic atomistic models by Monte Carlo simulations for joint X-ray and neutron-scattering curve fits, constrained by the requirement of correct disulfide bridges in the hinge, resulted in the determination of symmetric Y-shaped IgG2 structures. These molecular structures were distinct from those for asymmetric IgG1 and asymmetric and symmetric IgG4 and were attributable to the four hinge disulfides. Our IgG2 structures rationalize the existence of the human IgG1, IgG2, and IgG4 subclasses and explain the receptor-binding functions of IgG2., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Hui et al.)

The human IgG1 antibody subclass shows distinct properties compared with the IgG2, IgG3, and IgG4 subclasses and is the most exploited subclass in therapeutic antibodies. It is the most abundant subclass, has a half-life as long as that of IgG2 and IgG4, binds the FcγR receptor, and activates complement. There is limited structural information on full-length human IgG1 because of the challenges of crystallization. To rectify this, we have studied the solution structures of two human IgG1 6a and 19a monoclonal antibodies in different buffers at different temperatures. Analytical ultracentrifugation showed that both antibodies were predominantly monomeric, with sedimentation coefficients s20,w (0) of 6.3-6.4 S. Only a minor dimer peak was observed, and the amount was not dependent on buffer conditions. Solution scattering showed that the x-ray radius of gyration Rg increased with salt concentration, whereas the neutron Rg values remained unchanged with temperature. The x-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions were unchanged in different buffers to indicate conformational stability. Constrained atomistic scattering modeling revealed predominantly asymmetric solution structures for both antibodies with extended hinge structures. Both structures were similar to the only known crystal structure of full-length human IgG1. The Fab conformations in both structures were suitably positioned to permit the Fc region to bind readily to its FcγR and C1q ligands without steric clashes, unlike human IgG4. Our molecular models for human IgG1 explain its immune activities, and we discuss its stability and function for therapeutic applications., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Human IgG4 antibody shows therapeutically useful properties compared with the IgG1, IgG2, and IgG3 subclasses. Thus IgG4 does not activate complement and shows conformational variability. These properties are attributable to its hinge region, which is the shortest of the four IgG subclasses. Using high throughput scattering methods, we studied the solution structure of wild-type IgG4(Ser(222)) and a hinge mutant IgG4(Pro(222)) in different buffers and temperatures where the proline substitution suppresses the formation of half-antibody. Analytical ultracentrifugation showed that both IgG4 forms were principally monomeric with sedimentation coefficients s20,w(0) of 6.6-6.8 S. A monomer-dimer equilibrium was observed in heavy water buffer at low temperature. Scattering showed that the x-ray radius of gyration Rg was unchanged with concentration in 50-250 mm NaCl buffers, whereas the neutron Rg values showed a concentration-dependent increase as the temperature decreased in heavy water buffers. The distance distribution curves (P(r)) revealed two peaks, M1 and M2, that shifted below 2 mg/ml to indicate concentration-dependent IgG4 structures in addition to IgG4 dimer formation at high concentration in heavy water. Constrained x-ray and neutron scattering modeling revealed asymmetric solution structures for IgG4(Ser(222)) with extended hinge structures. The IgG4(Pro(222)) structure was similar. Both IgG4 structures showed that their Fab regions were positioned close enough to the Fc region to restrict C1q binding. Our new molecular models for IgG4 explain its inability to activate complement and clarify aspects of its stability and function for therapeutic applications., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Solution structures for antibodies are critical to understand function and therapeutic applications. The stability of the solution structure of rabbit IgG in different buffers and temperatures was determined by analytical ultracentrifugation and X-ray and neutron scattering. Rabbit IgG showed a principally monomeric species, which is well resolved from small amounts of a dimeric species. The proportion of dimer increased with increased concentration, decreased temperature and heavy water from 8% to 25% in all buffers except for high salt (250 mM NaCl). The Guinier X-ray radius of gyration R(G) likewise increased with concentration in 137 mM NaCl buffer but was unchanged in 250 mM NaCl buffer. The Guinier neutron R(G) values increased as the temperature decreased. The X-ray and neutron distance distribution curves P(r) revealed two peaks, M1 and M2, whose positions did not change with concentration to indicate unchanged structures under all these conditions. The maximum dimension increased with concentration because of dimer formation. Constrained scattering modeling reproducibly revealed very similar asymmetric solution structures for monomeric rabbit IgG in different buffers, in which the Fab-Fc and Fab-Fab pairs were separated by maximally extended hinge structures. The dimer was best modeled by two pairs of Fab regions forming tip-to-tip contacts. The intact rabbit IgG structures explained the ability of its two ligands, the Fc receptor and complement C1q, to bind to the top of its Fc region that is fully accessible and unhindered by the Fab regions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Of the four human IgG antibody subclasses IgG1-IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration R(G) increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab-Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab-Fc pairs change by 1-2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.

The capabilities of current computer simulations provide a unique opportunity to model small-angle scattering (SAS) data at the atomistic level, and to include other structural constraints ranging from molecular and atomistic energetics to crystallography, electron microscopy and NMR. This extends the capabilities of solution scattering and provides deeper insights into the physics and chemistry of the systems studied. Realizing this potential, however, requires integrating the experimental data with a new generation of modelling software. To achieve this, the CCP-SAS collaboration () is developing open-source, high-throughput and user-friendly software for the atomistic and coarse-grained molecular modelling of scattering data. Robust state-of-the-art molecular simulation engines and molecular dynamics and Monte Carlo force fields provide constraints to the solution structure inferred from the small-angle scattering data, which incorporates the known physical chemistry of the system. The implementation of this software suite involves a tiered approach in which GenApp provides the deployment infrastructure for running applications on both standard and high-performance computing hardware, and SASSIE provides a workflow framework into which modules can be plugged to prepare structures, carry out simulations, calculate theoretical scattering data and compare results with experimental data. GenApp produces the accessible web-based front end termed SASSIE-web, and GenApp and SASSIE also make community SAS codes available. Applications are illustrated by case studies: (i) inter-domain flexibility in two- to six-domain proteins as exemplified by HIV-1 Gag, MASP and ubiquitin; (ii) the hinge conformation in human IgG2 and IgA1 antibodies; (iii) the complex formed between a hexameric protein Hfq and mRNA; and (iv) synthetic `bottlebrush' polymers. [ABSTRACT FROM AUTHOR]