Penny A. Handford

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.

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Mutations in Fibrillin-1 Cause Congenital Scleroderma: Stiff Skin Syndrome

Stiff skin syndrome, an autosomal dominant congenital form of scleroderma, is caused by mutations in the domain of fibrillin-1 that mediates integrin binding. Variation in Rare Disease Linked to Common Skin Disorder In the epic words of British physician and researcher Dr. William Harvey, “Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by the careful investigation of cases of rarer forms of disease. For it has been found in almost all things, that what they contain of useful or of applicable nature, is hardly perceived unless we are deprived of them, or they become deranged in some way.” Such is the case for rare genetic diseases, which have provided the framework to understand some of the most devastating common diseases from just the simple permutation of a gene. Scleroderma, which literally means a pathological hardening of the skin, manifests as a complex phenotype. To better understand the etiology of scleroderma, Loeys and colleagues investigate four families with more than 10 affected individuals with a rare congenital form called stiff skin syndrome—which together account for more than 25% of the total cases currently documented in the literature—and identify the key mutations in a gene that encodes the connective tissue protein fibrillin-1. Fibrosis is not only seen in the context of systemic connective tissue disorders, but rather is often the major signature of the inflammatory burden in many common disorders, providing incentive to understand the factors critical in the initiation and maintenance of profibrotic programs. Mutations causing stiff skin syndrome cluster within a single domain of fibrillin-1 that mediates integin binding. Low amounts of an activated protein kinase that is triggered by ligand-integrin interactions provided further evidence for altered cell matrix interactions. This associates with evidence for activation of the TGFβ signaling cascade, a mechanism to instruct cells to deposit collagens and other matrix elements both during normal wound healing and in various fibrotic states. These data are consistent with a model in which integrins provide a means for cells to sample the matrix and to adjust their synthetic repertoire accordingly. Loss of this feedback would culminate in fibrosis. The results garnered from these patients with this rare disease prompted the authors to examine skin biopsies from five patients with common scleroderma, which surprisingly revealed that each scleroderma patient showed all of the abnormalities seen in stiff skin syndrome. These findings reinforce the power of studying rare genetic disorders to inform the underlying causes of common diseases, but also unveil a potential avenue for therapeutic intervention in common disorders that include fibrosis. The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence–encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor–β (TGFβ). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFβ concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

The first EGF-like domain from human factor IX contains a high-affinity calcium binding site.

It has been suggested that epidermal growth factor‐like (EGF‐like) domains, containing conserved carboxylate residues, are responsible for the high‐affinity calcium binding exhibited by a number of vitamin K‐dependent plasma proteins involved in the control of the blood coagulation cascade. These include the procoagulant factors IX and X, and the anticoagulants protein C and protein S. To test this hypothesis we have expressed the first EGF‐like domain from human factor IX (residues 46‐84) using a yeast secretion system, and examined calcium binding to the domain. Using 1H‐NMR to measure a calcium‐dependent shift assigned to Tyr69 we have detected a high‐affinity calcium binding site (Kd = 200‐300 microM). We suggest that other EGF‐like domains of this type may have similar calcium binding properties. In addition, we have completely assigned the aromatic region of the NMR spectrum by NOESY and COSY analysis, and have used these data to discuss the effect of calcium and pH on the conformation of the domain with reference to a model based on the structure of human EGF.

Mutations in the TGFβ Binding-Protein-Like Domain 5 of FBN1 Are Responsible for Acromicric and Geleophysic Dysplasias

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Fibrillin: from domain structure to supramolecular assembly

In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.

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.

Notch receptor–ligand binding and activation: Insights from molecular studies

Highlights ► We review the high resolution structures of the Notch receptor and ligands. ► Highlight the docking events of Notch receptor and ligand at the cell surface. ► Indicate the future challenges in understanding Notch receptor–ligand interactions.