Glycosaminoglycan-protein interactions and human complement factor H

Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association

\u3cem\u3eTrichodysplasia Spinulosa\u3c/em\u3e-Associated Polyomavirus Uses a Displaced Binding Site on VP1 to Engage Sialylated Glycolipids

Trichodysplasia spinulosa-associated Polyomavirus (TSPyV) was isolated from a patient suffering from trichodysplasia spinulosa, a skin disease that can appear in severely immunocompromised patients. While TSPyV is one of the five members of the polyomavirus family that are directly linked to a human disease, details about molecular recognition events, the viral entry pathway, and intracellular trafficking events during TSPyV infection remain unknown. Here we have used a structure-function approach to shed light on the first steps of TSPyV infection. We established by cell binding and pseudovirus infection studies that TSPyV interacts with sialic acids during attachment and/or entry. Subsequently, we solved high-resolution X-ray structures of the major capsid protein VP1 of TSPyV in complex with three different glycans, the branched GM1 glycan, and the linear trisaccharides α2,3- and α2,6-sialyllactose. The terminal sialic acid of all three glycans is engaged in a unique binding site on TSPyV VP1, which is positioned about 18 Å from established sialic acid binding sites of other polyomaviruses. Structure-based mutagenesis of sialic acid-binding residues leads to reduction in cell attachment and pseudovirus infection, demonstrating the physiological relevance of the TSPyV VP1-glycan interaction. Furthermore, treatments of cells with inhibitors of N-, O-linked glycosylation, and glycosphingolipid synthesis suggest that glycolipids play an important role during TSPyV infection. Our findings elucidate the first molecular recognition events of cellular infection with TSPyV and demonstrate that receptor recognition by polyomaviruses is highly variable not only in interactions with sialic acid itself, but also in the location of the binding site

Structural basis for complement factor H-linked age-related macular degeneration

This is the final version of the article. Available from the publisher via the DOI in this record.Nearly 50 million people worldwide suffer from age-related macular degeneration (AMD), which causes severe loss of central vision. A single-nucleotide polymorphism in the gene for the complement regulator factor H (FH), which causes a Tyr-to-His substitution at position 402, is linked to approximately 50% of attributable risks for AMD. We present the crystal structure of the region of FH containing the polymorphic amino acid His402 in complex with an analogue of the glycosaminoglycans (GAGs) that localize the complement regulator on the cell surface. The structure demonstrates direct coordination of ligand by the disease-associated polymorphic residue, providing a molecular explanation of the genetic observation. This glycan-binding site occupies the center of an extended interaction groove on the regulator's surface, implying multivalent binding of sulfated GAGs. This finding is confirmed by structure-based site-directed mutagenesis, nuclear magnetic resonance-monitored binding experiments performed for both H402 and Y402 variants with this and another model GAG, and analysis of an extended GAG-FH complex.B. Prosser is funded by the Wellcome Trust Structural Biology Training Program (075415/Z/04/Z). S. Johnson and P. Roversi were funded by grants to S.M. Lea from the Medical Research Council (MRC) of the United Kingdom (grants G0400389 and G0400775). D. Uhrin and P.N. Barlow were funded by the Wellcome Trust (078780/ Z/05/Z). S.J. Clark was funded by an MRC Doctoral Training Account (G78/7925), and R.B. Sim and A.J. Day were funded by MRC core funding to the MRC Immunochemistry Unit

Functional binding of hexanucleotides to 3C protease of hepatitis A virus

Oligonucleotides as short as 6 nt in length have been shown to bind specifically and tightly to proteins and affect their biological function. Yet, sparse structural data are available for corresponding complexes. Employing a recently developed hexanucleotide array, we identified hexadeoxyribonucleotides that bind specifically to the 3C protease of hepatitis A virus (HAV 3Cpro). Inhibition assays in vitro identified the hexanucleotide 5′-GGGGGT-3′ (G5T) as a 3Cpro protease inhibitor. Using 1H NMR spectroscopy, G5T was found to form a G-quadruplex, which might be considered as a minimal aptamer. With the help of 1H, 15N-HSQC experiments the binding site for G5T was located to the C-terminal β-barrel of HAV 3Cpro. Importantly, the highly conserved KFRDI motif, which has previously been identified as putative viral RNA binding site, is not part of the G5T-binding site, nor does G5T interfere with the binding of viral RNA. Our findings demonstrate that sequence-specific nucleic acid–protein interactions occur with oligonucleotides as small as hexanucleotides and suggest that these compounds may be of pharmaceutical relevance