Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40 in the tumorigenicity of human melanoma cells (Ledda

in the tumorigenicity of human melanoma cells (Ledda Erhard Hohenester1, Patrik Maurer2,3 and et al., 1997). Other activities of BM-40 include calciumRupert Timpl2 dependent binding to collagens and thrombospondin, bindDepartment of Crystallography, Birkbeck College, ing to platelet-derived growth factor (PDGF)-AB and London WC1E 7HX, UK and 2Abteilung Proteinchemie, -BB and the regulation of cell proliferation and matrix Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany metalloproteinase expression (Lane and Sage, 1994; 3Present address: Institut für Biochemie der Medizinischen Fakultät, Sage, 1997). Universität Köln, 50931 Köln, Germany The sequence of BM-40 has been highly conserved 1Corresponding author throughout vertebrate evolution (Lane and Sage, 1994), and a homologue with 38% identity to human BM-40 has BM-40 (also known as SPARC or osteonectin) is an also been found in the nematode C.elegans (Schwarzbauer anti-adhesive secreted glycoprotein involved in tissue and Spencer, 1993). The human protein consists of 286 remodelling. Apart from an acidic N-terminal segment, residues divided into three distinct domains: (i) an NBM-40 consists of a follistatin-like (FS) domain and terminal acidic segment (residues 1–52) binding several an EF-hand calcium-binding (EC) domain. Here we calcium ions with low affinity andmediating the interaction report the crystal structure at 3.1 Å resolution of the with hydroxyapatite; (ii) a follistatin-like (FS; Patthy and FS–EC domain pair of human BM-40. The two distinct Nikolics, 1993; Bork et al., 1996) domain (residues 53– domains interact through a small interface that involves 137) containing five disulfides and an N-linked complex the EF-hand pair of the EC domain. Residues impliccarbohydrate at Asn99; and (iii) an α-helical domain ated in cell binding, inhibition of cell spreading and containing two EF-hand calcium-binding sites (EC disassembly of focal adhesions cluster on one face of domain, residues 138–286;Maurer et al., 1995; Hohenester BM-40, opposite the binding epitope for collagens and et al., 1996). Pairs of FS and EC domains are found in a the N-linked carbohydrate. The elongated FS domain number of extracellular proteins, including SC1/hevin is structurally related to serine protease inhibitors of (Johnston et al., 1990; Girard and Springer, 1996), QR1 the Kazal family. Notable differences are an insertion (Guermah et al., 1991), testican (Alliel et al., 1993) into the inhibitory loop in BM-40 and a protruding and tsc-36 (Shibanuma et al., 1993). However, with the N-terminal β-hairpin with striking similarities to epiexception of hevin, which modulates high endothelial cell dermal growth factor. This hairpin is likely to act as adhesion to the basement membrane (Girard and Springer, a rigid spacer in proteins containing tandemly repeated 1996), the functions of proteins related to BM-40 are FS domains, such as follistatin and agrin, and forms unknown. the heparin-binding site in follistatin. We recently have solved the crystal structure at 2.0 Å