Calcium affinity, cooperativity, and domain interactions of extracellular EF-hands present in BM-40

The structure and function of cytosolic Ca2+-binding proteins containing EF -hands are well understood. Recently, the presence of EF-hands in an extrac ellular protein was for the first time proven by the structure determinatio n of the EC domain of BM-40 (SPARC (for secreted protein acidic and rich in cysteine)/osteonectin) (Hohenester, E., Maurer, P., Hohenadl, C., Timpl, R ., Jansonius, J. N., and Engel, J. (1996) Not. Struct. Biol. 3, 67-73). The structure revealed a pair of EF-hands with two bound Ca2+ ions. Two unusua l features were noted that distinguish the extracellular EF-hands of BM-40 from their cytosolic counterparts. An insertion of one amino acid into the loop of the first EF-hand causes a variant Ca2+ coordination, and a disulfi de bond connects the helices of the second EF-hand. Here we show that the e xtracellular EF-hands in the BM-40 EC domain bind Ca2+ cooperatively and wi th high affinity. The EC domain is thus in the Ca2+-saturated form in the e xtracellular matrix, and the EF-hands play a structural rather than a regul atory role. Deletion mutants demonstrate a strong interaction between the E C domain and the neighboring FS domain, which contributes about 10 kJ/mol t o the free energy of binding and influences cooperativity. This interaction is mainly between the FS domain and the variant EF-hand 1. Certain mutatio ns of Ca2+-coordinating residues changed affinity and cooperativity, but ot hers inhibited folding and secretion of the EC domain in a mammalian cell l ine. This points to a function of EF-hands in extracellular proteins during biosynthesis and processing in the endoplasmic reticulum or Golgi apparatu s.