The solution structure of the unbound IgG Fc receptor CD64 resembles its crystal structure: Implications for function

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

The solution structure of the unbound IgG Fc receptor CD64 resembles its crystal structure:Implications for function

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.</p

The Solution Structures of Two Human IgG1 Antibodies Show Conformational Stability and Accommodate Their C1q and FcγR Ligands.

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

Unravelling the interaction of the human IgG subclasses with their ligands using biophysical methods

Antibodies are responsible for binding to antigens through their Fab arms and eliciting an immune response by binding to Fcγ receptors (FcγR). IgG4 is associated with IgG4-related disease. Human IgG in blood consists of four subclasses (IgG1, IgG2, IgG3 and IgG4). In order to identify their solution structures, monoclonal IgG1 and IgG4 and myeloma IgG2 were studied using analytical ultracentrifugation (AUC) and small-angle X-ray and neutron scattering (SAXS, SANS), followed by atomistic modelling using molecular dynamics and Monte Carlo methods. The three IgG subclasses exhibit variable conformations and flexibility, reflecting their function. FcγRI (CD64) is the sole high-affinity human IgG receptor. Its crystal structure showed that its three domains D1, D2 and D3 form a compact “seahorse” shape. To identify its solution structure, AUC and SAXS data and their modelling showed that FcγRI is more elongated than its crystal structure, in which the D1 and D2 domains were no longer in contact with each other, and the D3 domain showed flexibility. The solution structures of the IgG1 and IgG4 complexes with FcγRI were determined by AUC, SAXS and SANS. These showed a 1:1 binding stoichiometry. Their atomistic modelling showed that the Fc region in IgG1/IgG4 accommodates one FcγRI asymmetrically, and the two Fab arms were displaced to enable FcγRI binding. The modelling showed that the bound FcγRI changed conformation back into a compact “seahorse” where D1 is folded back against the D2 domain. Here, the solution structure of full-length IgG with FcγRI resembled the crystal structures of the IgG Fc fragment complexes with FcγRI. It was concluded that the folding back of FcγRI into a “seahorse” conformation ensures 1:1 binding and high-affinity interaction, giving an effective and regulated immune response to remove foreign antigens. This thesis furthers our understanding of the conformations of human IgG and their association with disease

Characterisation of recombinant CD64.

(A) The elution of CD64 from an IgG sepharose affinity column (mAU, milli-absorbance units) (B) SDS-PAGE analyses of CD64. Lane 2 represents the non-reduced (NR) recombinant CD64 and Lane 3 represents reduced CD64 (R). The original SDS-PAGE image is shown in S1 Fig. Most of the CD64 was monomeric (arrowed as M) while small amounts of dimer CD64 are arrowed as D. (C) The LC-MS mass spectrum for recombinant CD64 is shown as a single peak at 32.00 kDa at a voltage of 350 V, together with a small peak at 64.01 kDa. (D) The theoretical charge states generated using MassHunter software are labelled. The three strongest peaks are expanded into the panel for clarity.</p

Experimental and modelled Kratky plots for CD64.

The normalised dimensionless Kratky plots for 0.11, 0.22, 0.33 and 0.44 mg/ml CD64 are shown as white, grey, dark grey and black circles respectively. The Kratky plots for each of the modelled X-ray curves for each CD64 concentration are shown as dotted, short dash, long dash and solid red lines respectively. The blue vertical line represents the point of inflection at a Q.RG value of 1.7.</p

The supporting information zip file provides the 100 best-fit models for CD64 corresponding to the fit searches of the X-ray curve at 0.11 mg/ml.

The supporting information zip file provides the 100 best-fit models for CD64 corresponding to the fit searches of the X-ray curve at 0.11 mg/ml.</p

Comparison of the crystal structures of CD64 with density plots of the 100 best-fit models of CD64.

(A-D) Ribbon diagrams of the four CD64 crystal structures correspond to unbound CD64 (PDB code 3RJD) and the three CD64-Fc co-crystals (PDB codes B, 4W4O, C, 4X4M and D, 4ZNE). The glycans in the crystal structures are shown as yellow sticks. (E-H) The density plots of the four sets of putative 100 best-fit CD64 models at four concentrations are shown. The energy-minimised starting structure of CD64 is shown as a black cartoon in the same views in all four panels. The 100 best-fit models were superimposed on the D2 domain. The volumes occupied by the D1, D2 and D3 domains in the 100 best-fit structures are represented as blue, orange and red wireframes respectively in each density plot. Two different views rotated by 90° of each density plot are displayed. In panel E, we also show a stick representation of 10 of the 100 best-fit CD64 models in a smaller scale to clarify how the density plots related to the atomistic models, with the best fit model shown as a blue ribbon.</p

X-ray scattering curve fits for the best-fit CD64 models.

The interpolated experimental X-ray scattering curves are indicated by open circles and the scattering curves of the best-fit models are indicated by blue continuous lines. The fits correspond to (A) 0.11 mg/ml, (B) 0.22 mg/ml, (C) 0.33 mg/ml and (D) 0.44 mg/ml of CD64 (Fig 4). The red lines represent the modelled curve for the energy-minimised starting structure. The yellow dashed lines represent the calculated scattering curve from the CD64 crystal structure (chain E, PDB code 4X4M). The insets represent the corresponding modelled P(r) curves (blue) overlaid onto the normalised experimental P(r) curves (black) from Fig 4F.</p