Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97-CD55.

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4.

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite-host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite-host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7-9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7-9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands.

Ca2+-dependent interface formation in fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common structural motif in extracellular and transmembrane proteins. K(d) values for Ca2+ vary from the millimolar to nanomolar range; however the molecular basis for this variation is poorly understood. We have measured K(d) values for six fibrillin-1 cbEGF domains, each preceded by a transforming growth factor beta-binding protein-like (TB) domain. Using NMR and titration with chromophoric chelators, we found that K(d) values varied by five orders of magnitude. Interdomain hydrophobic contacts between TB-cbEGF domains were studied by site-directed mutagenesis and could be correlated directly with Ca2+ affinity. Furthermore, in TB-cbEGF pairs that displayed high-affinity binding, NMR studies showed that TB-cbEGF interface formation was strongly Ca2+-dependent. We suggest that Ca2+ affinity is a measure of interface formation in both homologous and heterologous cbEGF domain pairs, thus providing a measure of flexibility in proteins with multiple cbEGF domains. These data highlight the versatile role of the cbEGF domain in fine tuning the regional flexibility of proteins and provide new constraints for the organization of fibrillin-1 within 10-12-nm microfibrils of the extracellular matrix.

Structural and functional properties of the human notch-1 ligand binding region.

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11-13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded beta sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region.

Crystallization and preliminary X-ray diffraction analysis of three EGF domains of EMR2, a 7TM immune-system molecule.

Crystals of three epidermal growth-factor-like (EGF) domains of EMR2 (143 residues) have been grown. EMR2 is a member of the EGF-TM7 family of proteins. Different splice variants exist with between three and five consecutive EGF modules linked to a seven-span transmembrane G-protein-coupled receptor. Although its precise function is unknown, EMR2 is highly expressed in immune tissues and has been shown to weakly bind CD55, a complement-system regulator. Here, crystallization of EMR2 in the presence of Ca(2+), Ba(2+) and Sr(2+) ions is reported. A complete data set has been collected from all three crystal types, all of which belong to space group P2(1). An anomalous Patterson map from the Ba(2+) crystal data reveals three Ba(2+) ions bound within the asymmetric unit.

Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization.

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.

Solution structure of the third TB domain from LTBP1 provides insight into assembly of the large latent complex that sequesters latent TGF-beta.

Almost all TGF-beta is secreted as part of a large latent complex. This complex is formed from three molecules, a latent transforming growth factor-beta binding protein (LTBP), which plays roles in targeting and activation, a latency associated peptide (LAP), which regulates latency, and the TGF-beta cytokine. LAP is the TGF-beta pro-peptide that is cleaved intracellularly prior to secretion, and TGF-beta binds non-covalently to LAP. Formation of the large latent complex is important for the efficient secretion of TGF-beta. Previous studies have revealed that the LTBP-LAP interaction is mediated by intracellular exchange of a single disulphide bond within the third, and only the third, TB domain (TB3) with LAP. We have previously reported the structure of a homologous TB domain from fibrillin-1. However, TB3 contains a two amino acid insertion, not found in fibrillin-1 TB domains, which is not amenable to molecular modelling. In order to clarify the basis of TB domain function, we have determined the solution NMR structure of TB3(LTBP1). Comparison with the fibrillin-1 TB domain reveals that the two-residue insertion is associated with a significant increase in solvent accessibility of one of the disulphide bonds (linking the second and sixth cysteine residues). Site-directed mutagenesis and NMR studies indicate that this is the only disulphide bond that can be removed without perturbing the TB domain fold. Furthermore, a ring of negatively charged residues has been identified that surrounds this disulphide bond. Homology modelling suggests that the surface properties of TB3 domains from different LTBP isoforms correlate with binding activities. This research provides testable hypotheses regarding the molecular basis of complex formation between LTBPs and LAPs.

Effects of the N2144S mutation on backbone dynamics of a TB-cbEGF domain pair from human fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) module and the transforming growth factor beta-binding protein-like (TB) module are the two major structural motifs found in fibrillin-1, the extracellular matrix (ECM) protein defective in the Marfan syndrome (MFS). An MFS-causing mutation, N2144S, which removes a calcium ligand in cbEGF32, does not detectably affect fibrillin-1 biosynthesis, rate of secretion, processing, or deposition of reducible fibrillin-1 into the ECM. Since the residue at position 2144 is normally engaged in calcium ligation, it is unable to mediate intermolecular interactions. We have shown previously that this mutation does not affect the folding properties of the TB or cbEGF domains in vitro, but does decrease calcium-binding in cbEGF and TB-cbEGF domain constructs. Here, we use NMR spectroscopy to probe the effects of the N2144S mutation on backbone dynamic properties of TB6-cbEGF32. Analysis of the backbone (15)N relaxation data of wild-type TB6-cbEGF32 has revealed a flexible inter-domain linkage. Parallel dynamics analysis of the N2144S mutant has shown increased flexibility in the region joining the two domains as well as in the calcium-binding site at the N terminus of cbEGF32. This research demonstrates that a small change in peptide backbone flexibility, which does not enhance proteolytic susceptibility of the domain pair, is associated with an MFS phenotype. Flexibility of the TB-cbEGF linkage is likely to contribute to the biomechanical properties of fibrillin-rich connective tissue microfibrils, and may play a role in the microfibril assembly process.