Gunasekar Susheel K, Asnani Mukta, Limbad Chandani, Haghpanah Jennifer S, Hom Wendy, Barra Hanna, Nanda Soumya, Lu Min, Montclare Jin Kim
Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, Brooklyn, New York 11201, USA.
Biochemistry. 2009 Sep 15;48(36):8559-67. doi: 10.1021/bi900534r.
The coiled-coil domain of cartilage oligomeric matrix protein (COMPcc) assembles into a homopentamer that naturally recognizes the small molecule 1,25-dihydroxyvitamin D(3) (vit D). To identify the residues critical for the structure, stability, oligomerization, and binding to vit D as well as two other small molecules, all-trans-retinol (ATR) and curcumin (CCM), here we perform an alanine scanning mutagenesis study. Ten residues lining the hydrophobic pocket of COMPcc were mutated into alanine; of the mutated residues, the N-terminal aliphatic residues L37, L44, V47, and L51 are responsible for maintaining the structure and function. Furthermore, two polar residues, T40 and Q54, within the N-terminal region when converted into alanine improve the alpha-helical structure, stability, and self-assembly behavior. Helical stability, oligomerization, and binding appear to be linked in a manner in which mutations that abolish helical structure and assembly bind poorly to vit D, ATR, and CCM. These results provide not only insight into COMPcc and its functional role but also useful guidelines for the design of stable, pentameric coiled-coils capable of selectively storing and delivering various small molecules.
软骨寡聚基质蛋白的卷曲螺旋结构域（COMPcc）组装成同源五聚体，该五聚体天然识别小分子1,25-二羟基维生素D(3)（维生素D）。为了确定对于结构、稳定性、寡聚化以及与维生素D以及另外两种小分子全反式视黄醇（ATR）和姜黄素（CCM）结合至关重要的残基，我们在此进行了丙氨酸扫描诱变研究。COMPcc疏水口袋内的10个残基被突变为丙氨酸；在这些突变残基中，N端脂肪族残基L37、L44、V47和L51负责维持结构和功能。此外，N端区域内的两个极性残基T40和Q54转化为丙氨酸后可改善α螺旋结构、稳定性和自组装行为。螺旋稳定性、寡聚化和结合似乎以一种方式相互关联，即消除螺旋结构和组装的突变与维生素D、ATR和CCM的结合很差。这些结果不仅为了解COMPcc及其功能作用提供了见解，也为设计能够选择性储存和递送各种小分子的稳定五聚体卷曲螺旋提供了有用的指导。
