Title : Crystal structure and mapping by site-directed mutagenesis of the collagen-binding
epitope of an activated
form of BM-40 /
SPARC /
osteonectin
Abstract :
- The extracellular calcium-binding domain ( positions 138-286) of the matrix protein BM-40 possesses a binding epitope of moderate affinity for several collagen types
- This epitope was predicted to reside in helix alphaA and to be partially masked by helix alphaC
- Here we show that deletion of helix alphaC produces a 10-fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix
- The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 A resolution
- This constitutively activated mutant was used to map the collagen-binding site following alanine mutagenesis at 13 positions
- Five residues were crucial for binding, R149 and N156 in helix alphaA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM-40
- These residues are spatially close and form a flat ring of 15 A diameter which matches the diameter of a triple-helical collagen domain
- The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens
- Selected mutations in the non-activated mutant DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40
Output (sent_index, trigger,
protein,
sugar,
site):
Output(Part-Of) (sent_index,
protein,
site):
- 0. form of BM-40, epitope
- 1. BM-40, domain
- 1. BM-40, epitope
- 1. BM-40, positions 138
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):