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Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

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 © 2011 Elsevier Inc. All rights reserved.)

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104-Glu1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RG values of 4.89-4.90 nm for C4b in 137-250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137-250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED-MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b. [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: 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]

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments dp6-dp24 corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6 - dp24 showed a small rotor speed dependence, where similar s20,w values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6 - dp24 gave radii of gyration RG values from 1.03 to 2.82 nm, cross-sectional radii of gyration RXS values from 0.31 to 0.65 nm, and maximum lengths L from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5,000 to 12,000 conformationally randomized HS structures gave best fit dp6 - dp24 molecular structures that were longer and more bent than their equivalents in heparin. Alternative fits were obtained for HS dp18 and dp24, indicating their higher bending and flexibility. We conclude that HS displays bent conformations that are significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin. [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]

The highly sulfated polysaccharides heparin and heparan sulfate (HS) play key roles in the regulation of physiological and pathophysiological processes. Despite its importance, no molecular structures of free HS have been reported up to now. By combining analytical ultracentrifugation, small angle x-ray scattering, and constrained scattering modeling recently used for heparin, we have analyzed the solution structures for eight purified HS fragments degree of polymerization 6-18 (dp6- dpl8) and dp24, corresponding to the predominantly unsulfated GIcA-GIcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s20,w of HS dp6 - dp24 showed a small rotor speed dependence, where similar 520,w values of 0.82-1.26 S (absorbance optics) and 1.05-1.34 S (interference optics) were determined. The corresponding x-ray scattering measurements of HS dp6 - dp24 gave radius of gyration (RG) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (RXS) values from 0.31 to 0.65 nm, and maximum lengths (L) from 3.0 to 10.0 nm. These data showed that HS has a longer and more bent structure than heparin. Constrained scattering modeling starting from 5000-8000 conformationally randomized HS structures gave best fit dp6 - dpl6 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dpl8 or dp24, indicating their higher flexibility. We conclude that HS displays an extended bent conformation that is significantly distinct from that for heparin. The difference is attributed to the different predominant monosaccharide sequence and reduced sulfation of HS, indicating that HS may interact differently with proteins compared with heparin. [ABSTRACT FROM AUTHOR]

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: 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]

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]

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]