B21-High Throughput SAXS
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Hannah F.
Bradford
,
Christophe J.
Lalaurie
,
Jayesh
Gor
,
Xin
Gao
,
Charis
Pericleous
,
Stephen J.
Perkins
,
Hannah
Britt
,
Konstantinos
Thalassinos
,
Ian
Giles
,
Anisur
Rahman
,
Mihaela
Delcea
,
Paul A.
Dalby
,
Thomas C. R.
Mcdonnell
Open Access
Abstract: Beta-2-Glycoprotein I (β2GPI) is the main autoantigenic target of antiphospholipid syndrome (APS) with antibodies leading to clinical manifestations. There are two known structural isomers of β2GPI, a J shape and a circular shaped one. The transition between these structures is incompletely understood, with the functional implications unknown. β2GPI is a substrate of the protease plasmin, which cleaves within the fifth domain of β2GPI leading to altered cellular binding. Very little is currently known regarding the structure and function of this protein variant. We present the first comprehensive structural characterisation plasmin-clipped β2GPI and the associated implications for pathogenic antibody binding to this protein. Methods: β2GPI was purified using an adapted acid-free process from healthy control plasma and cleaved with plasmin. Cleavage was confirmed by SDS-PAGE. Structural characterisation was undertaken using dynamic light scattering (DLS), small angle X-ray scattering (SAXS), ion mobility mass spectrometry (IMMS) and molecular dynamics simulation (MD). Activity was tested using inhibition of β2GPI ELISAs with patient samples and cleaved β2GPI in the fluid phase and cellular binding by flow cytometry using HUVEC cells. Results:
DLS revealed a significantly smaller hydrodynamic radius for plasmin-clipped β2GPI (p=0.0043). SAXS and MD analysis indicated a novel S-like structure of β2GPI only present in the plasmin-clipped sample whilst IMMS showed a different structure distributions in plasmin clipped compared to non-clipped B2GPI. The increased binding of autoantibodies was shown for plasmin-clipped β2GPI (p=0.056), implying a greater exposure of pathogenic epitopes following cleavage. Conclusions: Cleavage of β2GPI by plasmin results in the production of a unique S-shaped structural conformation and higher patient antibody binding. This novel structure may increase the production of antibodies and explain the loss of binding to phospholipids described previously for plasmin-clipped β2GPI.
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Feb 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[24505, 29178]
Open Access
Abstract: Human complement factor H (CFH) plays a central role in regulating activated C3b to protect host cells. CFH contain 20 short complement regulator (SCR) domains and eight N-glycosylation sites. The N-terminal SCR domains mediate C3b degradation while the C-terminal CFH domains bind to host cell surfaces to protect these. Our earlier study of Pichia-generated CFH fragments indicated a self-association site at SCR-17/18 that comprises a dimerization site for human factor H. Two N-linked glycans are located on SCR-17 and SCR-18. Here, when we expressed SCR-17/18 without glycans in an E. coli system, analytical ultracentrifugation showed that no dimers were now formed. To investigate this novel finding, full-length CFH and its C-terminal fragments were purified from human plasma and Pichia pastoris respectively, and their glycans were enzymatically removed using PNGase F. Using size-exclusion chromatography, mass spectrometry, and analytical ultracentrifugation, SCR-17/18 from Pichia showed notably less dimer formation without its glycans, confirming that the glycans are necessary for the formation of SCR-17/18 dimers. By surface plasmon resonance, affinity analyses interaction showed decreased binding of deglycosylated full-length CFH to immobilised C3b, showing that CFH glycosylation enhances the key CFH regulation of C3b. We conclude that our study revealed a significant new aspect of CFH regulation based on its glycosylation and its resulting dimerisation.
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Aug 2024
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18022]
Open Access
Abstract: 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.
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Apr 2024
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B21-High Throughput SAXS
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Open Access
Abstract: Heavy-chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities compared to conventional antibodies. To address the challenge of SARS-CoV-2, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody, and shows therapeutic efficacy in vivo. Here, we have characterised the solution arrangement of the molecule. Two 1,443 Da N-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier radius of gyration RG values, which provide information about structural elongation, were similar at 4.1-4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modelling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.
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Oct 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18022]
Open Access
Abstract: 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.
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Sep 2023
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[207821]
Open Access
Abstract: Collagen triple helices are critical in the function of mannan-binding lectin (MBL), an oligomeric recognition molecule in complement activation. The MBL collagen regions form complexes with the serine proteases MASP-1 and MASP-2 in order to activate complement, and mutations lead to common immunodeficiencies. To evaluate their structure-function properties, we studied the solution structures of four MBL-like collagen peptides. The thermal stability of the MBL collagen region was much reduced by the presence of a GQG interruption in the typical (X-Y-Gly)n repeat compared to controls. Experimental solution structural data were collected using analytical ultracentrifugation and small angle X-ray and neutron scattering. As controls, we included two standard Pro-Hyp-Gly collagen peptides (POG)10-13, as well as three more peptides with diverse (X-Y-Gly)n sequences that represented other collagen features. These data were quantitatively compared with atomistic linear collagen models derived from crystal structures and 12,000 conformations obtained from molecular dynamics (MD) simulations. All four MBL peptides were bent to varying degrees up to 85o in the best-fit MD models. The best-fit benchmark peptides (POG)n were more linear but exhibited a degree of conformational flexibility. The remaining three peptides showed mostly linear solution structures. In conclusion, the collagen helix is not strictly linear, the degree of flexibility in the triple helix depends on its sequence, and the triple helix with the GQG interruption showed a pronounced bend. The bend in MBL GQG peptides resembles the bend in the collagen of complement C1q and may be key for lectin pathway activation.
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Dec 2022
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[30822]
Open Access
Abstract: Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamic (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of BoNT/E. This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for BoNT/A. These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use.
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Jun 2022
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B21-High Throughput SAXS
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Open Access
Abstract: Human immunoglobulin 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 glycosylation sites in the Fc region is 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 5.82-6.29 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) of both forms of IgG3 showed a maximum length of 25-28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modelling 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 of the two forms 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 semi-rigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2 and IgG4.
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Jul 2021
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[18022]
Abstract: The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is comprised 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. Following deglycosylation using PNGase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass following glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. While the radius of gyration RG was unchanged, the cross-sectional radius of gyration, RXS-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 following deglycosylation. To explain these changes, atomistic scattering modelling 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, while the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the FcγR receptor in deglycosylated IgG1.
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Mar 2021
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[12342]
Abstract: Human zinc-α2-glycoprotein (ZAG) is a 42 kDa adipokine which regulates body fat mass and is associated with cachexia and obesity. ZAG belongs to the major histocompatibility complex class I protein family and binds long chain polyunsaturated fatty acids in its groove formed from the α1 and α2 domains. To identify the molecular basis of its lipid-binding function, we determined the first crystal structure at 2.49Å resolution for fatty acid-bound ZAG, where the ligand was the fluorescent 11-(dansylamino)undecanoic acid (DAUDA). The 192 kDa crystallographic asymmetric unit contained six ZAG and eight fatty acid molecules in unique conformations. Six fatty acid molecules were localised to the ZAG grooves, where its tails were bound in two distinct conformations. The carboxylate groups of three fatty acids projected out of the groove, while the fourth was hydrogen bonded with R73 inside the groove. Other ligand-residue contacts were primarily hydrophobic. A new fatty acid site was revealed for two further DAUDA molecules at the ZAG α3 domains. Following conformational changes from unbound ZAG, the α3 domains formed tetrameric β-barrel structures lined by fatty acid molecules that doubled the binding capacity of ZAG. Analytical ultracentrifugation revealed that ZAG in solution was a monomer in the absence of DAUDA, but formed small amounts of tetramers with DAUDA. By showing that ZAG binds fatty acids in different locations, we demonstrate an augmented mechanism for fatty acid binding in ZAG that is distinct from other known fatty acid binding proteins, and may be relevant to cachexia.
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Sep 2019
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