The C-terminal portion of BM-40 (SPARC/osteonectin) is an autonomously folding and crystallisable domain that binds calcium and collagen IV.
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 (QR1, 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 delta I) and the calcium-binding EC domain alone (mutant delta I, 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 delta I, II could be readily crystallized and diffraction patterns with a resolution limit of 2.4 A resolution were obtained. Calcium binding to this fragment was ten times weaker (Kd = 0.8 microM) than for the wild-type protein. Identical reversible increases in alpha-helicity upon calcium binding were observed for the 150-residue long mutant delta I, 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 Kd 2.4 mM at 1 microM, to Kd 0.3 mM at 100 microM 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 (Kd = 19 microM) only sixfold lower than that of intact human BM-40.
Maurer
P
P
Abt. Biophysikalische Chemie, Biozentrum University, Basel, Switzerland.
Hohenadl
C
C
Hohenester
E
E
Göhring
W
W
Timpl
R
R
Engel
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002942:Circular Dichroism; D003094:Collagen / Q000378:metabolism*; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015676:Osteonectin / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D017931:DNA Primers; D015676:Osteonectin; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D002118:Calcium