Structural insights into antibody-mediated mucosal immunity.

The mucosal regions of the body are responsible for defense against environmental pathogens. Particularly in the lumen of the gut, antibody-mediated immune responses are critical for preventing invasion by pathogens. In this chapter, we review structural studies that have illuminated various aspects of mucosal immunity. Crystal structures of IgA1-Fc and IgA-binding fragments of the polymeric immunoglobulin receptor and Fc alphaRI, combined with models of intact IgA and IgM from solution scattering studies, reveal potential mechanisms for immune exclusion and induction of inflammatory responses. Other recent structures yield insights into bacterial mechanisms for evasion of the host immune response.

Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.

Loss of fibroblast growth factor receptor 2 ligand-binding specificity in Apert syndrome.

Craniosynostosis syndromes are autosomal dominant human skeletal diseases that result from various mutations in fibroblast growth factor receptor genes (Fgfrs). Apert syndrome (AS) is one of the most severe craniosynostosis syndromes and is associated with severe syndactyly of the hands and feet and with central nervous system malformations. AS is caused by specific missense mutations in one of two adjacent amino acid residues (S252W or P253R) in the highly conserved region linking Ig-like domains II and III of FGFR2. Here we demonstrate that these mutations break one of the cardinal rules governing ligand specificity of FGFR2. We show that the S252W mutation allows the mesenchymal splice form of FGFR2 (FGFR2c) to bind and to be activated by the mesenchymally expressed ligands FGF7 or FGF10 and the epithelial splice form of FGFR2 (FGFR2b) to be activated by FGF2, FGF6, and FGF9. These data demonstrate loss of ligand specificity of FGFR2 with retained ligand dependence for receptor activation. These data suggest that the severe phenotypes of AS likely result from ectopic ligand-dependent activation of FGFR2.

Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system.

The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.

New insights into heparin-induced FGF oligomerization.

Fibroblast growth factors (FGFs) play important roles in a variety of developmental processes in mammals. The dependence of their activity on heparin binding has been a puzzle that, in recent years, has been the subject of active investigation. Recent structural analyses on complexes of FGFs with heparin fragments or heparin analogs have unveiled the extreme complexity of these systems.

Heparin-induced self-association of fibroblast growth factor-2. Evidence for two oligomerization processes.

Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF-2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.

Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity.