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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 well-defined oligomers with purified heparin fragments (equivalent to S-domains), and indicated a dissociation constant K(d) of approximately 0.5 μM. Unchanged FH structures that are bivalently cross-linked at SCR-7 and SCR-20 with heparin explained the sedimentation coefficients of the FH-heparin oligomers. The X-ray radius of gyration, R(G), 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 full-length FH gave K(d) values of 1-3 μM, and similar but weaker K(d) 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.

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-dp18) and dp24, corresponding to the predominantly unsulfated GlcA-GlcNAc domains of heparan sulfate. Unlike heparin, the sedimentation coefficient s(20,)(w) of HS dp6-dp24 showed a small rotor speed dependence, where similar s(20,)(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 (R(G)) values from 1.03 to 2.82 nm, cross-sectional radius of gyration (R(XS)) 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-dp16 molecular structures that were longer and more bent than their equivalents in heparin. No fits were obtained for HS dp18 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.

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 (phi) and psi (psi) 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 2009 Elsevier Ltd. All rights reserved.)

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 microM 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.

Factor H (FH) of the complement system acts as a regulatory cofactor for the factor I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-16 to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic fibroblast growth factor-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney., (Copyright 2002 Academic Press.)

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]

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]

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]

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients. [ABSTRACT FROM AUTHOR]