Sasaki T, Hohenester E, Göhring W, Timpl R
Max-Planck-Institut für Biochemie, D-82152, Martinsried, Germany.
EMBO J. 1998 Mar 16;17(6):1625-34. doi: 10.1093/emboj/17.6.1625.
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.
基质蛋白BM-40的细胞外钙结合结构域（第138 - 286位）对几种胶原蛋白类型具有中等亲和力的结合表位。该表位预计位于αA螺旋中，并部分被αC螺旋掩盖。在此我们表明，删除αC螺旋会使胶原蛋白亲和力增加10倍，类似于该螺旋经蛋白水解切割后的情况。在2.8埃分辨率下，突变体的晶体结构清楚地证明了预测的空间位阻限制的消除。这个组成型激活的突变体用于在13个位置进行丙氨酸诱变后绘制胶原蛋白结合位点。五个残基对结合至关重要，αA螺旋中的R149和N156，以及连接BM-40两个EF手的环区域中的L242、M245和E246。这些残基在空间上接近，形成一个直径为15埃的扁平环，与三螺旋胶原蛋白结构域的直径相匹配。这些突变对与I型和IV型胶原蛋白结合的影响相似，表明两种胶原蛋白上的结合位点几乎相同。非激活突变体DeltaI中的选定突变也降低了胶原蛋白结合，这与表位位于相同位置但在完整的BM-40中以更隐蔽的形式存在一致。
