Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy

2010 ◽  
Vol 432 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Yuki Abe ◽  
Jayesh Gor ◽  
Daniel G. Bracewell ◽  
Stephen J. Perkins ◽  
Paul A. Dalby

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 RG 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.

2020 ◽  
Vol 295 (48) ◽  
pp. 16342-16358
Author(s):  
Nilufar Kadkhodayi-Kholghi ◽  
Jayesh S. Bhatt ◽  
Jayesh Gor ◽  
Lindsay C. McDermott ◽  
Daniel P. Gale ◽  
...  

The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (Rg) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy.


Immunobiology ◽  
2016 ◽  
Vol 221 (10) ◽  
pp. 1223
Author(s):  
Nilufar Kadkhodayi-Kholghi ◽  
Jayesh Gor ◽  
Anna Ferlin ◽  
Lindsay C. McDermott ◽  
Daniel P. Gale ◽  
...  

2019 ◽  
Vol 294 (28) ◽  
pp. 10789-10806 ◽  
Author(s):  
Gar Kay Hui ◽  
Antoni D. Gardener ◽  
Halima Begum ◽  
Charles Eldrid ◽  
Konstantinos Thalassinos ◽  
...  

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 (s20, w0) 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 (Rg) is 5.2–5.4 nm, after allowing for radiation damage at higher concentrations, and that the neutron Rg 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.


2012 ◽  
Vol 56 (4) ◽  
pp. 1899-1906 ◽  
Author(s):  
Shane J. Caldwell ◽  
Albert M. Berghuis

ABSTRACTAminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage.


1989 ◽  
Vol 261 (3) ◽  
pp. 1043-1046 ◽  
Author(s):  
E J Wachtel ◽  
T Sverbilova ◽  
W D McCubbin ◽  
C M Kay

The solution structure of troponin C from turkey skeletal muscle was studied at low pH by small-angle X-ray-scattering. We find that troponin C at pH 5.3 in the presence of Mg2+ has a triaxial radius of gyration and maximum dimension comparable with those of the crystallized protein. However, the relative disposition of domains is more similar to that found for the highly homologous rabbit protein in solution at pH 7.4.


2009 ◽  
Vol 131 (2) ◽  
pp. 502-508 ◽  
Author(s):  
Morten Christensen ◽  
Kristoffer Haldrup ◽  
Klaus Bechgaard ◽  
Robert Feidenhans’l ◽  
Qingyu Kong ◽  
...  

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