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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. [ABSTRACT FROM AUTHOR]

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris , the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease. [ABSTRACT FROM AUTHOR]

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was compared with the blocked (POG)10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4-17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease. [ABSTRACT FROM AUTHOR]

Native IgA1, for which no crystal structure is known, contains an O-galactosylated 23-residue hinge region that joins its Fab and Fc regions. IgA nephropathy (IgAN) is a leading cause of chronic kidney disease in developed countries. Because IgA1 in IgAN often has a poorly O-galactosylated hinge region, the solution structures of monomeric IgA1 from a healthy subject and three IgAN patients with four different O-galactosylation levels were studied. Analytical ultracentrifugation showed that all four IgA1 samples were monomeric with similar sedimentation coefficients, s0 20,w. X-ray scattering showed that the radius of gyration (Rg) slightly increased with IgA1 concentration, indicating self-association, although their distance distribution curves, P(r), were unchanged with concentration. Neutron scattering indicated similar Rg values and P(r) curves, although IgA1 showed a propensity to aggregate in heavy water buffer. A new atomistic modelling procedure based on comparisons with 177000 conformationally-randomized IgA1 structures with the individual experimental scattering curves revealed similar extended Y-shaped solution structures for all four differentiallyglycosylated IgA1 molecules. The final models indicated that the N-glycans at Asn263 were folded back against the Fc surface, the C-terminal tailpiece conformations were undefined and hinge O-galactosylation had little effect on the solution structure. The solution structures for full-length IgA1 showed extended hinges and the Fab and Fc regions were positioned asymmetrically to provide ample space for the functionally-important binding of two FcaR receptors to its Fc region. Whereas no link between O-galactosylation and the IgA1 solution structure was detected, an increase in IgA1 aggregation with reduced O-galactosylation may relate to IgAN. [ABSTRACT FROM AUTHOR]

Complement factor H (CFH) is the major regulator of the central complement protein C3b in the alternative pathway of complement activation. A molecular view of the CFH interaction with native heparan sulfate (HS) is central for understanding the mechanism of how surface-bound CFH interacts with C3b bound to host cell surfaces. HS is composed of sulfated heparin-like S-regions that alternate with desulfated NA-regions. Solution structural studies of heparin (equivalent to the S-regions) and desulfated HS (the NA-regions) by scattering and ultracentrifugation showed that each structure was mostly extended and partially bent, but with greater bending and flexibility in the NA-regions compared to the S-regions. Their solution structures have been deposited in the Protein Data Bank. The largest HS oligosaccharides showed more bent and flexible structures than those for heparin. A folded-back domain structure for the solution structure of the 20 domains in CFH was determined likewise. CFH binds to the S-regions but less so to the NA-regions of HS. The bivalent interaction of CFH-heparin was observed by ultracentrifugation, and binding studies of CFH fragments with heparin-coated sensor chips. In common with other CFH interactions with its physiological and pathophysiological ligands, the CFH-heparin and CFH-C3b interactions have moderate micromolar dissociation constants KD, meaning that these complexes do not fully form in vivo. The combination of the solution structures and binding studies indicated a two-site interaction model of CFH with heparin at cell surfaces. By this, the bivalent binding of CFH to a cell surface is co-operative. Defective interactions at either of the two independent CFH-heparin sites reduce the CFH interaction with surface-bound C3b and lead to immune disorders. [ABSTRACT FROM AUTHOR]

FH (Factor H) with 20 SCR (short complement regulator) domains is a major serum regulator of complement, and genetic defects in this are associated with inflammatory diseases. Heparan sulfate is a cell-surface glycosaminoglycan composed of sulfated S-domains and unsulfated NA-domains. To elucidate the molecular mechanism of binding of FH to glycosaminoglycans, we performed ultracentrifugation, X-ray scattering and surface plasmon resonance with FH and glycosaminoglycan fragments. Ultracentrifugation showed that FH formed up to 63% of welldefined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constantKd of approximately 0.5 µM. Unchanged FH structures that are bivalently crosslinked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, RG, of FH in the presence of heparin fragments 18-36 monosaccharide units long increased significantly from 10.4 to 11.7 nm, and the maximum lengths of FH increased from 35 to 40 nm, confirming that large compact oligomers had formed. Surface plasmon resonance of immobilized heparin with fulllength FH gave Kd values of 1-3 µM, and similar but weaker Kd values of 4-20 µM for the SCR-6/8 and SCR-16/20 fragments, confirming co-operativity between the two binding sites. The use of minimally-sulfated heparan sulfate fragments that correspond largely to NA-domains showed much weaker binding, proving the importance of S-domains for this interaction. This bivalent and co-operative model of FH binding to heparan sulfate provides novel insights on the immune function of FH at host cell surfaces. [ABSTRACT FROM AUTHOR]

Abstract: Factor H (FH) is the major plasma regulator of the central complement protein C3b in the alternative pathway of complement activation. The elucidation of the FH interactions with five major ligands (below) is complicated by their weak μM dissociation constants K D and FH multivalency. We present the first survey of all the K D values for the major FH–ligand interactions and critically review their physiological significance. [(i)] FH self-association is presently well-established. We review multiple data sets that show that 5–14% of FH is self-associated in physiological conditions. FH self-association is significant for both laboratory investigations and physiological function. [(ii)] The FH–C3b complex shows low μM affinity, meaning that the complex is not fully formed in plasma. In addition, C3, its hydrolysed form C3u, and its cleaved forms C3b and C3d show multimerisation. Current data favour a model when two C3b molecules bind independently to one FH molecule, as opposed to a 1:1 stoichiometry where FH wraps itself around C3b. [(iii)] Heparin is often used as an analogue of the polyanionic host cell surface. The FH–heparin complex also shows a low μM affinity, again meaning that complexes are not fully formed in vivo. The oligomeric FH–heparin complexes clarify a two-site interaction model of FH with host-cell surfaces. [(iv)] Reinvestigation of the FH and C-reactive protein (CRP) interaction revealed that this can only occur in plasma when CRP levels are elevated during acute-phase conditions. Given that CRP binds more weakly to the His402 allotype of FH than the Tyr402 allotype, this suggested a link with age-related macular degeneration (AMD). [(v)] FH activity is inhibited by zinc, which causes FH to aggregate strongly. High levels of bioavailable zinc occur in sub-retinal pigment epithelial deposits which lead to AMD. Excess zinc binds weakly to a central region of FH, explaining how zinc inhibits FH regulation of C3b. [Copyright &y& Elsevier]

The study of the relationship between the complex structures and numerous physiological functions of the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS) has continued to thrive in the past decade. Though it is clear that the monosaccharide sequences of these polysaccharides must determine their ability to modulate the action of growth factors, morphogens, chemokines, cytokines, and many other extracellular proteins, the exact details of this dependence still prove elusive. Sequence determines the 3D structure of GAGs at more than one level; detailed sequences of highly sulfated regions may influence affinity for specific proteins in some cases, but in addition attention has been called to the importance of the length and spacing of these highly sulfated sequences, which are separated by unsulfated domains. Within the sulfated "S-domains", the internal dynamics of the conformationally flexible iduronate pyranose ring have continued to interest NMR spectroscopists and molecular modelers. New studies of the relative degrees of flexibility of sulfated and unsulfated domains lead to an overall model of heparin/HS in which protein-binding, highly sulfated S-domains with well-defined conformations are separated by more flexible NA-domains. [ABSTRACT FROM AUTHOR]

Abstract: Analytical ultracentrifugation and solution scattering provide different multi-parameter structural and compositional information on proteins. The joint application of the two methods supplements high resolution structural studies by crystallography and NMR. We summarise the procedures required to obtain equivalent ultracentrifugation and X-ray and neutron scattering data. The constrained modelling of ultracentrifugation and scattering data is important to confirm the experimental data analysis and yields families of best-fit molecular models for comparison with crystallography and NMR structures. This modelling of ultracentrifugation and scattering data is described in terms of starting models, their conformational randomisation in trial-and-error fits, and the identification of the final best-fit models. Seven applications of these methods are described to illustrate the current state-of-the-art. These include the determination of antibody solution structures (the human IgG4 subclass, and oligomeric forms of human IgA and its secretory component), the solution structures of the complement proteins of innate immunity (Factor H and C3/C3u) and their interactions with macromolecular ligands (C-reactive protein), and anionic polysaccharides (heparin). Complementary features of joint ultracentrifugation and scattering experiments facilitate an improved understanding of crystal structures (illustrated for C3/C3u, C-reactive protein and heparin). If a large protein or its complex cannot be crystallised, the joint ultracentrifugation-scattering approach provides a means to obtain an overall macromolecular structure. [Copyright &y& Elsevier]

Many immunologically relevant proteins possess multidomain structures. Molecular structures both at the level of the individual domain and that of the intact protein are required for a full appreciation of function and control. Two recently develop structural approaches are reviewed here. Analogy modelling methods are based on the current understanding of many protein structures, and make possible the identification of folds for superfamilies of unknown structures. An integrated multidisciplinary predictive approach has been successfully applied to the von Willebrand factor type A, proteoglycan tandem repeat and factor I/membrane attack complex domains. The available experimental and predictive evidence is assembled in order to identify a known three-dimensional structure related to the unknown one of interest. Neutron and X-ray scattering curve modelling provides information on the full multidomain structure in solution. As scattering curves can be calculated from known atomic structures, the present availability of structures for many domains in conjunction with tight constraints based on these structures and the covalent connections between them results in a small family of allowed best-fit structures for a given scattering curve. The curve-fit procedure can be automated, and whole multidomain structures can be determined to a positional precision of the order of 0.2-1 nm. Such models are informative on the steric accessibility of each domain and their functional activity, and this is illustrated for antibody, cell-surface and complement proteins. [ABSTRACT FROM AUTHOR]

Heavy chain–only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARSCoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus 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 characterized the solution arrangement of the molecule. Two 1443 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 RG values, which provide information about structural elongation, were similar at 4.1 to 4.2 nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling 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. [ABSTRACT FROM AUTHOR]

Abstract: Secretory component (SC) in association with polymeric IgA (pIgA) forms secretory IgA (SIgA), the major antibody active at mucosal surfaces. SC also exists in a free form in secretions, with innate neutralizing properties against important pathogens. IgA-bound SC and free secretory component (FSC) are both produced by proteolytic cleavage of the polymeric Ig receptor whose function is to transport IgA and IgM across mucosal epithelia. Although the proteases have not been characterised and the site(s) of cleavage of the polymeric Ig receptor has been debated, it has been assumed that bound and free SC are produced by cleavage at the same site. Here we show by SDS-PAGE analyses that FSC is slightly smaller than SIgA1- or SIgA2-bound SC when purified simultaneously. The FSC preparation was functionally active, shown by binding to dimeric and polymeric IgA, and by its ability to trigger a respiratory burst by binding to ‘SC receptors’ on eosinophils. We also show that FSC from different human secretions have different molecular sizes. The solution structure of FSC from colostrum was studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of 4.25S is close to that for recombinant FSC. The X-ray scattering curve showed that FSC adopts a compact structure in solution which corresponds well to the J-shaped domain arrangement determined previously for recombinant FSC which terminates at residue Arg585. The smaller sizes of the FSC forms are attributable to variable cleavages of the C-terminal linker region, and may result from the absence of dimeric IgA. The FSC modelling accounts for the lack of effect of the C-terminal linker on the known functions of FSC. [Copyright &y& Elsevier]

Abstract: Factor H (FH) is a major regulator of complement alternative pathway activation. It is composed of 20 short complement regulator (SCR) domains and is genetically associated as a risk factor for age-related macular degeneration. Previous studies on FH suggested that it existed in monomeric and dimeric forms. Improved X-ray scattering and analytical ultracentrifugation methodology for wild-type FH permitted a clarification of these oligomeric properties. Data at lower concentrations revealed a dependence of the X-ray radius of gyration values on concentration that corresponded to the weak self-association of FH. Global sedimentation equilibrium fits indicated that a monomer–dimer equilibrium best described the data up to 1.3 mg/ml with a fitted dissociation constant K D of 28 μM and that higher oligomers formed at increased concentrations. The K D showed that about 85–95% of serum FH will be monomeric in the absence of other factors. Size-distribution analyses in sedimentation velocity experiments showed that monomeric FH was the major species but that as many as six oligomeric forms co-existed with it. The data were explained in terms of two weak dimerisation sites recently identified in the SCR-6/8 and SCR-16/20 fragments of FH with similar K D values. These observations indicate a mechanism for the progressive self-association of FH and may be relevant for complement regulation and the formation of drusen deposits that are associated with age-related macular degeneration. [Copyright &y& Elsevier]

Properdin regulates the alternative pathway of the complement system of immune defence by stabilising the C3 convertase complex. It contains six thrombospondin repeat type I (TSR-1 to TSR-6) domains and an N-terminal domain. Properdin exists as either a dimer, trimer or tetramer. In order to determine the solution structure of multiple TSR domains, the molecular structures of dimeric and trimeric properdin were studied by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that the dimer and trimer have radii of gyration RG values of 7.5nm and 10.3nm, respectively, and cross-sectional radii of gyration RXS values of 1.3nm and 1.5nm, respectively. Distance distribution functions showed that the maximum lengths of the dimer and trimer were 25nm and 30nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 5.1S and 5.2S for the dimer and trimer forms, respectively. Homology models for the TSR domains were constructed using the crystal structure of the TSP-2 and TSP-3 domains in human thrombospondin as templates. Properdin could be represented by seven TSR domains, not six as believed, since the crystal structure determined for TSP-2 and TSP-3 showed that the N-terminal domain (TSR-0) could be represented by a truncated TSR domain with the same six conserved Cys residues found in TSR-1 to TSR-6. Automated constrained molecular modelling revealed the solution conformations of multiple TSR domains in properdin at medium resolution. The comparison of 3125 systematically generated conformational models for the trimer with the X-ray data showed that good curve fits could be obtained by assuming that the linker between adjacent TSR domains possessed limited flexibility. Good trimer models correspond to partially collapsed triangular structures, and extended triangular shapes do not fit the data. The corresponding 3125 models for the dimer revealed a similar outcome in which a partially collapsed TSR structure gave good fits. The models account for the effect of mutations that cause properdin deficiencies, and suggest that the biologically active TSR-4, TSR-5 and TSR-6 domains are exposed for protein–protein interactions. The role of the other TSR domains in properdin may be to act as spacers to make TSR-4, TSR-5 and TSR-6 accessible for function. [Copyright &y& Elsevier]

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigenbinding 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 s0 20,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. [ABSTRACT FROM AUTHOR]

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 5.56 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. Thismodel 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. [ABSTRACT FROM AUTHOR]

Abstract Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states—notably amyloids—have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle x-ray scattering (SAXS), atomistic molecular dynamic simulations, single-molecule Förster resonance energy transfer, and aggregation-prone region predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, R g , of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full MD simulations from the same conditions and located the conformational changes in the native state to the constant domain of the light chain (C L). This C L displacement was independently confirmed using single-molecule Förster resonance energy transfer measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted APR, suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation. Graphical Abstract Unlabelled Image Highlights • Elucidation of local changes in native conformation that promote protein aggregation • Unprecedented resolution of native-like conformers by fitting MD simulations to SAXS data • smFRET of dual-labeled Fab A33 confirms conformational changes. • Local unfolding of Fab A33 exposes a predicted aggregation-prone region. [ABSTRACT FROM AUTHOR]

Abstract: 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 (250mM NaCl). The Guinier X-ray radius of gyration R G likewise increased with concentration in 137mM NaCl buffer but was unchanged in 250mM 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 &y& Elsevier]

Abstract: A novel therapeutic reagent TT30 was designed to be effective in diseases of the alternative pathway of complement such as paroxysmal nocturnal hemoglobinuria and other diseases. TT30 is constructed from the first four short complement regulator (SCR) domains of complement receptor type 2 (CR2) that bind to complement C3d, followed by the first five SCR domains of complement factor H that bind to complement C3b. In order to assess how TT30 binds to C3d and C3b, we determined the TT30 solution structure by a combination of analytical ultracentrifugation, X-ray scattering and constrained modeling. The sedimentation coefficients and radius of gyration of TT30 were unaffected by citrate or phosphate-buffered saline buffers and indicate an elongated monomeric structure with a sedimentation coefficient of 3.1 S and a radius of gyration R G of 6.9 nm. Molecular modeling starting from 3000 randomized TT30 conformations showed that high-quality X-ray curve fits were obtained with extended SCR arrangements, showing that TT30 has a limited degree of inter-SCR flexibility in its solution structure. The best-fit TT30 structural models are readily merged with the crystal structure of C3b to show that the four CR2 domains extend freely into solution when the five complement factor H domains are bound within C3b. We reevaluated the solution structure of the CR2–C3d complex that confirmed its recent crystal structure. This recent CR2–C3d crystal structure showed that TT30 is able to interact readily with C3d ligands in many orientations when TT30 is bound to C3b. [Copyright &y& Elsevier]

Abstract: The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at >10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein–protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD. [Copyright &y& Elsevier]

Abstract: The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin–protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6–dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6–dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6–dp36 gave radii of gyration R G that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9–15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin–protein complexes revealed similar ranges of phi (φ) and psi (ψ) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes. [Copyright &y& Elsevier]

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca2+-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mri Ca2+ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaC1 concentration. The sedimentation coefficients of pentameric and decameric CRP were 6.4 Sand in excess of 7.6 S, respectively. In the absence of Ca2+, CRP partially dissociates into its protomers and the NaC1 concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration RG values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged KD value of 21 LM in solution (140 mrwi NaCI, 2 mri Ca2+) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar KD value of 23 x M (140 mrwt NaCl, 2 mri Ca2+), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mri Ca2+ and 140 mri NaCI are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response. [ABSTRACT FROM AUTHOR]

Factor H (FH) regulates the activation of C3b in the alternative complement pathway, both in serum and at host cell surfaces. It is composed of 20 short complement regulator (SCR) domains. The Y402H polymorphism in FH is a risk factor for age-related macular degeneration. C-reactive protein (CRP) is an acute phase protein that binds Ca2+. We established the FHCRP interaction using improved analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and synchrotron x-ray scattering methods. Physiological FH and CRP concentrations were used in 137 mrt NaC1 and 2 mri Ca2+, in which the occurrence of denatured CRP was avoided. In solution, AUC revealed FH-CRP binding. The FH-CRP interaction inhibited the formation of higher FH oligomers, indicating that CRP blocked FH dimerization sites at both SCR-6/8 and SCR-16/20. SPR confirmed the FH-CRP interaction and its NaCl concentration dependence upon using either immobilized FH or CRP. The SCR-115 fragment of FH did not bind to CRP. In order of increasing affinity, SCR-16120, SCR-6/8(His-402), and SCR-618 (Tyr-402) fragments bound to CRP. X-ray scattering showed that FH became more compact when binding to CRP, which is consistent with CRP binding at two different FH sites. We concluded that FH and CRP bind at elevated acute phase concentrations of CRP in physiological buffer. The SCR-16/20 site is novel and indicates the importance of the FH-CRP interaction for both age-related macular degeneration and atypical hemolytic uremic syndrome. [ABSTRACT FROM AUTHOR]

Abstract: Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K D 1 of 2.6 μM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d. [Copyright &y& Elsevier]

Abstract: Factor H (FH), a major serum regulator of C3b in the complement alternative pathway, is composed of 20 short complement regulator (SCR) domains. Earlier solution structures for FH showed that this has a folded-back domain arrangement and exists as oligomers. To clarify the molecular basis for this, analytical ultracentrifugation and X-ray scattering studies of native FH were performed as a function of NaCl concentration and pH. The sedimentation coefficient for the FH monomer decreased from 5.7 S to 5.3 S with increase in NaCl concentration, showing that weak electrostatic inter-domain interactions affect its folded-back structure. FH became more elongated at pH 9.4, showing the involvement of histidine residue(s) in its folded-back structure. Similar studies of partially deglycosylated FH suggested that oligosaccharides were not significant in determining the FH domain structure. The formation of FH oligomers decreased with increased NaCl concentration, indicating that electrostatic interactions also affect this. X-ray scattering showed that the maximum length of FH increased from 32 nm in low salt to 38 nm in high salt. Constrained X-ray scattering modelling was used to generate significantly improved FH molecular structures at medium resolution. In 50 mM NaCl, the modelled structures showed that inter-SCR domain contacts are likely, while these contacts are fewer in 250 mM NaCl. The results of this study show that the conformation of FH is affected by its local environment, and this may be important for its interactions with C3b and when bound to polyanionic cell surfaces. [Copyright &y& Elsevier]

Abstract: Polymorphisms in factor H (FH), a major regulator of complement activation, and the accumulation of high zinc concentrations in the outer retina are both associated with age-related macular degeneration. FH is inhibited by zinc, which causes FH to aggregate. To investigate this, we quantitatively studied zinc-induced FH self-association by X-ray scattering and analytical ultracentrifugation to demonstrate uncontrolled FH oligomerisation in conditions corresponding to physiological levels of FH and pathological levels of zinc in the outer retina. By scattering, FH at 2.8–7.0 μM was unaffected until [Zn] increased to 20 μM, whereupon the radius of gyration, R G, values increased from 9 to 15 nm at [Zn]=200 μM. The maximum dimension of FH increased from 32 to 50 nm, indicating that compact oligomers had formed. By ultracentrifugation, size-distribution analyses showed that monomeric FH at 5.57 S was the major species at [Zn] up to 60 μM. At [Zn] above 60 μM, a series of large oligomers were formed, ranging up to 100 S in size. Oligomerisation was reversed by ethylenediaminetetraacetic acid. Structurally distinct large oligomers were observed for Cu, while Ni, Cd and Fe showed low amounts of oligomers and Mg and Ca showed no change. Fluid-phase assays showed reduced FH activities that correlated with increased oligomer formation. The results were attributed to different degrees of stabilisation of weak self-dimerisation sites in FH by transition metals. The relevance of metal-induced FH oligomer formation to complement regulation and age-related macular degeneration is discussed. [Copyright &y& Elsevier]

Abstract: Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B-cells through its binding to C3d, a cleavage fragment of the major complement component C3. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains in a partially folded-back but flexible structure. Here, the effect of C3d binding to CR2 was determined by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient of unbound CR2 is 4.03 S in 50 mM NaCl. Because this agrees well with a value of 3.93 S in 137 mM NaCl, the overall CR2 structure is unaffected by change in ionic strength. Unbound C3d exists in monomer-dimer and monomer-trimer equilibria in 50 mM NaCl, but as a monomer only in 137 mM NaCl. In c(s) size-distribution analyses, an equimolar mixture of the CR2–C3d complex in 50 mM NaCl revealed a single peak shifted to 4.52 S when compared to unbound CR2 at 4.03 S to show that the complex had formed. The CR2–C3d complex in 137 mM NaCl showed two peaks at 2.52 S and 4.07 S to show that this had dissociated. Solution structural models for the CR2 SCR-1/2 complex with C3d and CR2 SCR-1/15 were superimposed. These gave an average sedimentation coefficient of 4.57 S for the complex, in good agreement with the observed value of 4.52 S. It is concluded that CR2 does not detectably change conformation when C3d is bound to it. Consistent with previous analyses, its C3d complex is not formed in physiological salt conditions. The implications of these solution results for its immune role are discussed. To our knowledge, this is the first solution structural study of a large multidomain SCR protein CR2 bound to its physiological ligand C3d. [Copyright &y& Elsevier]

Abstract: Factor H (FH) is a plasma glycoprotein that plays a central role in regulation of the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and heparin. X-ray scattering and analytical ultracentrifugation showed that SCR-1/5 was monomeric, while SCR-16/20 formed dimers. The Guinier radius of gyration R G of 4.3 nm for SCR-1/5 and those of 4.7 nm and about 7.8 nm for monomeric and dimeric SCR-16/20, respectively, showed that their structures are partially folded back and bent. The distance distribution function P(r) showed that SCR-1/5 has a maximum dimension of 15 nm while monomeric and dimeric SCR-16/20 are 17 nm and about 27 nm long, respectively. The sedimentation coefficient of 2.4 S for SCR-1/5 showed no concentration-dependence, while that for SCR-16/20 was 2.8 S for the monomer and 3.9 S for the dimer. Sedimentation equilibrium data showed that SCR-1/5 is monomeric while SCR-16/20 exhibited a weak monomer–dimer equilibrium with a dissociation constant of 16 μM. The constrained scattering and sedimentation modelling of SCR-1/5 and SCR-16/20 showed that partially folded-back and bent flexible SCR arrangements fitted both data sets better than extended linear arrangements, and that the dimer was best modelled in the SCR-16/20 model by an end-to-end association of two SCR-20 domains. The SCR-1/5 and SCR-16/20 models were conformationally similar to the previously determined partially folded-back structure for intact wild-type FH, hence suggesting a partial explanation of the intact FH structure. Comparison of the SCR-16/20 model with the crystal structure of C3b clarified reasons for the distribution of mutations leading to atypical haemolytic uraemic syndrome. [Copyright &y& Elsevier]

Abstract: Human complement receptor type 1 (CR1, CD35) is a type I membrane-bound glycoprotein that belongs to the regulators of complement activity (RCA) family. The extra-cellular component of CR1 is comprised of 30 short complement regulator (SCR) domains, whereas complement receptor type 2 (CR2) has 15 SCR domains and factor H (FH) has 20 SCR domains. The domain arrangement of a soluble form of CR1 (sCR1) was studied by X-ray scattering and analytical ultracentrifugation. The radius of gyration R G of sCR1 of 13.4(±1.1) nm is not much greater than those for CR2 and FH, and its R G /R 0 anisotropy ratio is 3.76, compared to ratios of 3.67 for FH and 4.1 for CR2. Unlike CR2, but similar to FH, two cross-sectional R G ranges were identified that gave R XS values of 4.7(±0.2) nm and 1.2(±0.7) nm, respectively, showing that the SCR domains adopt a range of conformations including folded-back ones. The distance distribution function P(r) showed that the most commonly occurring distance in sCR1 is at 11.5 nm. Its maximum length of 55 nm is less than double those for CR2 or FH, even though sCR1 has twice the number of SCR domains compared to CR2 Sedimentation equilibrium experiments gave a mean molecular weight of 235 kDa for sCR1. This is consistent with the value of 245 kDa calculated from its composition including 14 N-linked oligosaccharide sites, and confirmed that sCR1 is a monomer in solution. Sedimentation velocity experiments gave a sedimentation coefficient of 5.8 S. From this, the frictional ratio (f/f 0) of sCR1 was calculated to be 2.29, which is greater than those of 1.96 for CR2 and 1.77 for FH. The constrained scattering modelling of the sCR1 solution structure starting from homologous SCR domain structures generated 5000 trial conformationally randomised models, 43 of which gave good scattering fits to show that sCR1 has a partly folded-back structure. We conclude that the inter-SCR linkers show structural features in common with those in FH, but differ from those in CR2, and the SCR arrangement in CR1 will permit C3b or C4b to access all three ligand sites. [Copyright &y& Elsevier]

Abstract: Factor H (FH) is a major complement control protein in serum. The seventh short complement regulator (SCR-7) domain of the 20 in FH is associated with age-related macular degeneration through a Tyr402His polymorphism. The recombinant SCR-6/8 domains containing either His402 or Tyr402 and their complexes with a heparin decasaccharide were studied by analytical ultracentrifugation and X-ray scattering. The sedimentation coefficient is concentration dependent, giving a value of 2.0 S at zero concentration and a frictional ratio f/f o of 1.2 for both allotypes. The His402 allotype showed a slightly greater self-association than the Tyr402 allotype, and small amounts of dimeric SCR-6/8 were found for both allotypes in 50 mM, 137 mM and 250 mM NaCl buffers. Sedimentation equilibrium data were interpreted in terms of a monomer–dimer equilibrium with a dissociation constant of 40 μM for the His402 form. The Guinier radius of gyration R G of 3.1–3.3 nm and the R G /R O ratio of 2.0–2.1 showed that SCR-6/8 is relatively extended in solution. The distance distribution function P(r) showed a maximum dimension of 10 nm, which is less than the length expected for a linear domain arrangement. The constrained scattering and sedimentation modelling of FH SCR-6/8 showed that bent SCR arrangements fit the data better than linear arrangements. Previously identified heparin-binding residues were exposed on the outside curvature of this bent domain structure. Heparin caused the formation of a more linear structure, possibly by binding to residues in the linker. It was concluded that the His402 allotype may self-associate more readily than the Tyr402 allotype, SCR-6/8 is partly responsible for the folded-back structure of intact FH, and SCR-6/8 changes conformation upon heparin binding. [Copyright &y& Elsevier]

Abstract: Complement receptor type 2 (CR2, CD21) is a cell surface protein that links the innate and adaptive immune response during the activation of B cells. The extracellular portion of CR2 comprises 15 or 16 short complement regulator (SCR) domains, for which the overall arrangement in solution is unknown. This was determined by constrained scattering and ultracentrifugation modelling. The radius of gyration of CR2 SCR 1-15 was determined to be 11.5 nm by both X-ray and neutron scattering, and that of its cross-section was 1.8 nm. The distance distribution function P(r) showed that the overall length of CR2 SCR 1-15 was 38 nm. Sedimentation equilibrium curve fits gave a mean molecular weight of 135,000 (±13,000) Da, in agreement with a fully glycosylated structure. Velocity experiments using the g*(s) derivative method gave a sedimentation coefficient of 4.2 (±0.1) S. In order to construct a model of CR2 SCR 1-15 for constrained fitting, homology models for the 15 SCR domains were combined with randomised linker peptides generated by molecular dynamics simulations. Using an automated procedure, the analysis of 15,000 possible CR2 SCR 1-15 models showed that only those models in which the 15 SCR domains were flexible but partially folded back accounted for the scattering and sedimentation data. The best-fit CR2 models provided a visual explanation for the versatile interaction of CR2 with four ligands C3d, CD23, gp350 and IFN-α. The flexible location of CR2 SCR 1-2 is likely to facilitate interactions of C3d–antigen complexes with the B cell receptor. [Copyright &y& Elsevier]

Immunoglobulin A (IgA) is unique amongst antibodies in being able to form polymeric structures that may possess important functions in the pathology of specific diseases. IgA also forms complexes with other plasma proteins, the IgA1-human serum albumin (HSA) complex (IgA1-HSA) being typical. We have purified this complex using a novel two-step purification based on thiophilic chromatography and gel filtration, and characterised this. HSA is linked covalently to the tailpiece of IgA1 by a disulphide bond between Cys471 in IgA1 and Cys34 in HSA. IgA1-HSA binds to IgA receptors on neutrophils and monocytes, and elicits a respiratory burst that is comparable in magnitude to that of monomeric IgA1. The solution arrangement of IgA1-HSA was identified by X-ray scattering and ultracentrifugation. The radius of gyration R G of 7.5(±0.3)nm showed that IgA1-HSA is more extended in solution than IgA1 (R G of 6.1–6.2nm). Its distance distribution function P(r) showed two peaks that indicated a well-separated solution structure similar to that for IgA1, and a maximum dimension of 25nm, which is greater than that of 21nm for IgA1. Sedimentation equilibrium showed that the IgA1:HSA stoichiometry is 1:1. Sedimentation velocity resulted in a sedimentation coefficient of 6.4S and a frictional ratio of 1.87, which is greater than that of 1.56 for IgA1. The constrained modelling of the IgA1-HSA structure using known structures for IgA1 and HSA generated 2432 conformationally randomised models of which 52 gave good scattering fits. The HSA structure was located at the base of the Fc fragment in IgA1 in an extended arrangement. Such a structure accounts for the functional activity of IgA1-HSA, and supports our previous modelling analysis of the IgA1 solution structure. The IgA1-HSA complex may suggest the potential for creating a new class of targeted therapeutic reagents based on the coupling of IgA1 to carrier proteins. [Copyright &y& Elsevier]

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 17nm. The molecular mass of CR2-Ig ranged between 101,000Da and 107,000Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600Da. 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. [Copyright &y& Elsevier]

Human immunoglobulin D (IgD) occurs most abundantly as a membrane-bound antibody on the surface of mature B cells (mIgD). IgD possesses the longest hinge sequence of all the human antibody isotypes, with 64 residues connecting the Fab and Fc fragments. A novel rapid purification scheme of secreted IgD from the serum of an IgD myeloma patient using thiophilic (T-gel) and lectin affinity chromatography gave a stable, homogeneous IgD preparation. Synchrotron X-ray scattering and analytical ultracentrifugation of IgD identified the solution arrangement of its Fab and Fc fragments, and thereby its hinge structure. The Guinier X-ray radius of gyration R G of 6.9(±0.1)nm showed that IgD is more extended in solution than the immunoglobulin subclass IgA1 (R G of 6.1–6.2nm). Its distance distribution function P(r) showed a single peak at 4.7nm and a maximum dimension of 23nm. Velocity experiments gave a sedimentation coefficient of 6.3S, which is similar to that for IgA1 at 6.2S. The complete IgD structure was modelled using molecular dynamics to generate IgD hinge structures, to which homology models for the Fab and Fc fragments were connected. Good scattering curve fits were obtained with 18 semi-extended best fit IgD models that were filtered from 8500 trial models. These best-fit models showed that the IgD hinge does not correspond to an extended polypeptide structure. The averaged solution structure arrangement of the Fab and Fc fragments in IgD is principally T-shaped and flexible, with contribution from Y-shaped and inverted Y-shaped structures. Although the linear sequence of the IgD hinge is much longer, comparison with previous scattering modelling of IgA1 and IgA2(m)1 suggests that the hinge of IgA1 and IgD are more similar than might have been expected, Both possess flexible T-shaped solution structures, probably reflecting the presence of restraining O-linked sugars. [Copyright &y& Elsevier]

Kallmann''s syndrome corresponds to a loss of sense of smell and hypogonadotrophic hypogonadism. Defects in anosmin-1 result in the X-linked inherited form of Kallmann''s syndrome. Anosmin-1 is an extracellular matrix protein comprised of an N-terminal, cysteine-rich (Cys-box) domain and a whey acidic protein-like (WAP) domain, followed by four fibronectin type III (FnIII) domains. The solution structures of recombinant proteins containing the first three domains (PIWF1) and all six domains (PIWF4) were determined by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that PIWF1 and PIWF4 have different radii of gyration (R G) values of 3.1nm and 6.7nm, respectively, but similar cross-sectional radii of gyration (R XS) values of 1.5nm and 1.9nm, respectively. Distance distribution functions showed that the maximum lengths of PIWF1 and PIWF4 were 11nm and 23nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 2.52S and 3.55S for PIWF1 and PIWF4, respectively. The interpretation of the scattering data by constrained modelling requires homology models for all six domains in anosmin-1. While models were already available for the WAP and FnIII domains, searches suggested the Cys-box domain may resemble the cysteine-rich region of the insulin-like growth factor receptor. Automated constrained molecular modelling based on joining the anosmin-1 domains with structurally randomised linkers resulted in 10,000 models for anosmin-1. A trial-and-error search showed that about 0.1–1.4% of these models fitted the X-ray data. The best models showed that the three domains and six domains in PIWF1 and PIWF4, respectively, were extended. The inter-domain linkers in anosmin-1 could not all be extended at the same time, and there was evidence for inter-domain flexibility. Models with folded-back domain arrangements do not fit the data. These solution structures account for the known biological function of anosmin-1, in particular its ability to interact with its three macromolecular ligands. [Copyright &y& Elsevier]

Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, a fragment of C3, the major complement component. Previous crystal structures of the C3d–CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed that the two SCR domains of CR2 form contact with each other in a closed V-shaped structure. SCR 1 and SCR 2 are connected by an unusually long eight-residue linker peptide. Medium-resolution solution structures for CR2 SCR 1-2, C3d, and their complex were determined by X-ray scattering and analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR 1-2, its radius of gyration R G of 2.12(±0.05)nm, its maximum length of 10nm and its sedimentation coefficient of 1.40(±0.03)S do not agree with those calculated from the crystal structures, and instead suggest an open structure. Computer modelling of the CR2 SCR1-2 solution structure was based on the structural randomisation of the eight-residue linker peptide joining SCR 1 and SCR 2 to give 9950 trial models. Comparisons with the X-ray scattering curve indicated that the most favoured arrangements for the two SCR domains corresponded to an open V-shaped structure with no contacts between the SCR domains. For C3d, X-ray scattering and sedimentation velocity experiments showed that it exists as a monomer–dimer equilibrium with a dissociation constant of 40μM. The X-ray scattering curve for monomeric C3d gave an R G value of 1.95nm, and this together with its value of 3.17S gave good agreement with the monomeric C3d crystal structure. Modelling of the C3d dimer gave good agreements with its scattering and ultracentrifugation parameters. For the complex, scattering and ultracentrifugation experiments showed that there was no dimerisation, indicating that the C3d dimerisation site was located close to the CR2 SCR 1-2 binding site. The R G value of 2.44(±0.1)nm, its length of 9nm and its value of 3.45(±0.01)S showed that its structure was not much more elongated than that of C3d. Calculations with 9950 models of CR2 SCR 1-2 bound to C3d through SCR 2 showed that SCR 1 formed an open V-shaped structure with SCR 2 and was capable of interacting with the surface of C3d. We conclude that the open V-shaped structures formed by CR2 SCR 1-2, both when free and when bound to C3d, are optimal for the formation of a tight two-domain interaction with its ligand C3d. [Copyright &y& Elsevier]

Immunoglobulin A (IgA), the most abundant human immunoglobulin, mediates immune protection at mucosal surfaces as well as in plasma. It exists as two subclasses IgA1 and IgA2, and IgA2 is found in at least two allotypic forms, IgA2m(1) or IgA2m(2). Compared to IgA1, IgA2 has a much shorter hinge region, which joins the two Fab and one Fc fragments. In order to assess its solution structure, monomeric recombinant IgA2m(1) was studied by X-ray and neutron scattering. Its Guinier X-ray radius of gyration RG is 5.18 nm and its neutron RG is 5.03 nm, both of which are significantly smaller than those for monomeric IgA1 at 6.1–6.2 nm. The distance distribution function P(r) for IgA2m(1) showed a broad peak with a subpeak and gave a maximum dimension of 17 nm, in contrast to the P(r) curve for IgA1, which showed two distinct peaks and a maximum dimension of 21 nm. The sedimentation coefficients of IgA1 and IgA2m(1) were 6.2 S and 6.4 S, respectively. These data show that the solution structure of IgA2m(1) is significantly more compact than IgA1. The complete monomeric IgA2m(1) structure was modelled using molecular dynamics to generate random IgA2 hinge structures, to which homology models for the Fab and Fc fragments were connected to generate 10,000 full models. A total of 104 compact best-fit IgA2m(1) models gave good curve fits. These best-fit models were modified by linking the two Fab light chains with a disulphide bridge that is found in IgA2m(1), and subjecting these to energy refinement to optimise this linkage. The averaged solution structure of the arrangement of the Fab and Fc fragments in IgA2m(1) was found to be predominantly T-shaped and flexible, but also included Y-shaped structures. The IgA2 models show full steric access to the two FcαRI-binding sites at the Cα2–Cα3 interdomain region in the Fc fragment. Since previous scattering modelling had shown that IgA1 also possessed a flexible T-shaped solution structure, such a T-shape may be common to both IgA1 and IgA2. The final models suggest that the combination of the more compact IgA2m(1) and the more extended IgA1 structures will enable human IgA to access a broader range of antigens than either acting alone. The hinges of both IgA subclasses appear to show reduced flexibility when compared to their equivalents in IgG, and this may be important for maintaining an extended IgA structure. [Copyright &y& Elsevier]

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 RG of rCrry was determined to be 4.9–5.0 nm, and the RG of the cross-section was 1.2–1.5 nm as determined by X-ray and neutron scattering. The RG of mCrry-Ig was 6.6–6.7 nm, and the RG 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 2.4 S for rCrry and 5.4 S 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. [Copyright &y& Elsevier]