The C-terminal Portion of BM-40 (SPARC/Osteonectin) is an Autonomously Folding and Crystallisable Domain that Binds Calcium and Collagen IV

Abstract

The extracellular glycoprotein BM-40 consists of three domains, an acidic domain I, a follistatin (FS)-like domain II and a calcium-binding EC domain with an EF-hand related motif. BM-40 and several other related proteins (OR1, SC1/hevin, testican and tsc-36/FRP) are members of a novel modular protein family that share the FS domain followed by an EC domain. We have expressed this pair of FS and EC domains (mutant ΔI) and the calcium-binding EC domain alone (mutant ΔI,II) of human BM-40 as recombinant proteins in human 293 cells. Circular dichroism demonstrated that both mutants were obtained as folded proteins with a distinct three-dimensional conformation. In addition, mutant ΔI,II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 Å resolution were obtained. Calcium binding to this fragment was ten times weaker ( K d= 0.8 μM) than for the wild-type protein. Identical reversible increases in α-helicity upon calcium binding were observed for the 150-residue long mutant ΔI,II and for BM-40 (286 residues). A 26-residue synthetic peptide corresponding to the EF-hand related motif exhibited much weaker calcium binding. The apparent dissociation constant decreased with increasing peptide concentration (from K d2.4 mM at 1 μM, to K d0.3 mM at 100 μM peptide concentration) and calcium binding was accompanied by dimerization of the peptide. This suggests that for strong calcium binding the EF-hand related motif has to be embedded into a larger protein domain that can form an autonomously folding protein module. The EC domain was also shown by surface plasmon resonance assay to be responsible for calcium-dependent binding to collagen IV with an affinity ( K d= 19 μM) only sixfold lower than that of intact human BM-40.