Vroni Knott

Solution structure of a pair of calcium-binding epidermal growth factor-like domains: implications for the Marfan syndrome and other genetic disorders.

The nuclear magnetic resonance structure of a covalently linked pair of calcium-binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, the protein defective in the Marfan syndrome, is described. The two domains are in a rigid, rod-like arrangement, stabilized by interdomain calcium binding and hydrophobic interactions. We propose a model for the arrangement of fibrillin monomers in microfibrils that reconciles structural and antibody binding data, and we describe a set of disease-causing mutations that provide the first clues to the specificity of cbEFG interactions. The residues involved in stabilizing the domain linkage are highly conserved in fibrillin, fibulin, thrombomodulin, and the low density lipoprotein receptor. We propose that the relative orientation of tandem cbEGF domains in these proteins is similar, but that in others, including Notch, pairs adopt a completely different conformation.

The structure of a Ca(2+)-binding epidermal growth factor-like domain: its role in protein-protein interactions.

Abstract Various diverse extracellular proteins possess Ca 2+ -binding epidermal growth factor (EGF)-like domains, the function of which remains uncertain. We have determined, at high resolution (1.5 A), the crystal structure of such a domain, from human clotting factor IX, as a complex with Ca 2+ . The Ca 2+ ligands form a classic pentagonal bipyramid with six ligands contributed by one polypeptide chain and the seventh supplied by a neighboring EGF-like domain. The crystal structure identifies the role of Ca 2+ in maintaining the conformation of the N-terminal region of the domain, but more importantly demonstrates that Ca 2+ can directly mediate protein-protein contacts. The observed crystal packing of the domains provides a plausible model for the association of multiple tandemly linked EGF-like domains in proteins such as fibrillin-1, Notch, and protein S. This model is consistent with the known functional data and suggests a general biological role for these domains.

Solution structure of the transforming growth factor beta-binding protein-like module, a domain associated with matrix fibrils.

Here we describe the high resolution nuclear magnetic resonance (NMR) structure of a transforming growth factor β (TGF‐β)‐binding protein‐like (TB) domain, which comes from human fibrillin‐1, the protein defective in the Marfan syndrome (MFS). This domain is found in fibrillins and latent TGF‐β‐binding proteins (LTBPs) which are localized to fibrillar structures in the extracellular matrix. The TB domain manifests a novel fold which is globular and comprises six antiparallel β‐strands and two α‐helices. An unusual cysteine triplet conserved in the sequences of TB domains is localized to the hydrophobic core, at the C‐terminus of an α‐helix. The structure is stabilized by four disulfide bonds which pair in a 1–3, 2–6, 4–7, 5–8 pattern, two of which are solvent exposed. Analyses of MFS‐causing mutations and the fibrillin‐1 cell‐binding RGD site provide the first clues to the surface specificity of TB domain interactions. Modelling of a homologous TB domain from LTBP‐1 (residues 1018–1080) suggests that hydrophobic contacts may play a role in its interaction with the TGF‐β1 latency‐associated peptide.

Molecular Analysis of the Epidermal Growth Factor-like Short Consensus Repeat Domain-mediated Protein-Protein Interactions DISSECTION OF THE CD97-CD55 COMPLEX

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 μm) and rapid off-rate (at least 0.6 s−1). The interaction is Ca2+ -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with aK D at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.

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-γ 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.

Solution Structure of the LDL Receptor EGF-AB Pair: A Paradigm for the Assembly of Tandem Calcium Binding EGF Domains

BACKGROUND From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

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. Kd values for Ca2+ vary from the millimolar to nanomolar range; however the molecular basis for this variation is poorly understood. We have measured Kd values for six fibrillin-1 cbEGF domains, each preceded by a transforming growth factor β-binding protein-like (TB) domain. Using NMR and titration with chromophoric chelators, we found that Kd 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.

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.

Crystallization of a calcium-binding EGF-like domain

Crystals of a calcium-binding epidermal growth factor (EGF)-like domain of human clotting factor IX suitable for X-ray diffraction analysis have been obtained by vapour diffusion (sitting drop) against 48% PEG 400. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit-cell dimensions a = b = 40.3, c = 98.2 A. The crystals diffract beyond 1.5 A resolution and are relatively stable in the X-ray beam. This is the first reported crystallization of a calcium-binding EGF-like domain.