Key residues involved in calcium-binding motifs in EGF-like domains

Abstract

Many extracellular proteins with diverse functions contain domains similar to epidermal growth factor (EGF), a number of which have a consensus Asp/Asn, Asp/Asn, Asp*/Asn*, Tyr/Phe (where the asterisk denotes a beta-hydroxylated residue). These include the coagulation factors IX and X, proteins with two EGF-like domains, the first of which contains the consensus residues. The first EGF-like domain of human factor IX contains a calcium-binding site, which is believed to be responsible for one of the high-affinity sites detected in this protein. Similar results have been obtained for bovine factor X. We have now used protein engineering and 1H-NMR techniques to investigate the importance of individual consensus residues for ligand binding. Measurement of a calcium-dependent Tyr 69 shift in the isolated first EGF-like domain from human factor IX demonstrates that Asp 47, Asp 49, and Asp 64 are directly involved in this binding. Gln 50, whose importance has previously been overlooked, is also involved in this binding. Two mutations in this domain, Asp 47----Glu, and Asp 64----Asn, present in patients with haemophilia B, reduce calcium binding to the domain greater than 4-fold and greater than 1,000-fold, respectively. Furthermore, the defective calcium binding of Asn 64 can be partially rescued by the compensatory mutation Gln 50----Glu. This latter mutation, when introduced singly more than doubles the affinity of the domain for calcium. This study thus defines residues involved in a new type of calcium-binding site and provides strong circumstantial evidence for calcium-binding motifs in many extracellular proteins, including the developmentally important proteins of Drosophila, notch, delta and crumbs.

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

“…Five EGF-like domains are present (Appella et al, 1988;Campbell and Bork, 1993): three are found in tandem (aa 147±189, 190±236 and 237±290) whereas two other domains present in a less conserved form are also identified in the same region, overlapping with the others (aa 118±159 and 263±343). None of these domains contains the laminintype or calcium-binding EGF-like domain signature (Engel, 1989;Handford et al, 1991). In addition, a stretch of amino acids located at position 83±129 obeys the chitin binding consensus described for (b1-4)-N-acetyl-D-glucosamine polymer specific lectins and plant chitinases (Wright et al, 1991).…”

New gene, nel, encoding a Mr 93 K protein with EGF‐like repeats is strongly expressed in neural tissues of early stage chick embryos

“…, is present at the head of some EGF-like structures. The nel protein has the consensus sequence of the type I1 Ca2+ binding site (Handford et al, 1991) at the head of the second and fifth EGF-like structures. The presumed cytoplasmic domain of the nel protein is a cysteine rich structure similar to the C-terminal structure of the adhesion factor von Willebrand factor and does not have the functionally catalytic sequence such as that of kinases.…”

Solution Structure and Dynamics of a Calcium Binding Epidermal Growth Factor-like Domain Pair from the Neonatal Region of Human Fibrillin-1

“…A subset of these domains contains a calcium binding (cb) consensus sequence, i.e. (D/N)X(D/N)(E/Q)X m (D/ N)*X n (Y/F) (where m and n are variable, and * indicates a potential ␤-hydroxylation site) (3)(4)(5) (Fig. 1a).…”