Ochoa Wendy F, Torrecillas Alejandro, Fita Ignacio, Verdaguer Nuria, Corbalán-García Senena, Gomez-Fernandez Juan C
Instituto de Biología Molecular de Barcelona (CSIC), Jordi Girona Salgado 18-26, E-08034 Barcelona, Spain.
Biochemistry. 2003 Jul 29;42(29):8774-9. doi: 10.1021/bi034713g.
Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.
蛋白激酶C（α）（PKC（α））是一种调节细胞生理及其生长、分化和凋亡的关键酶。已知全反式维甲酸（atRA）可调节PKC活性，但其作用机制甚至与PKC的可能结合方式均未明确。PKC（α）的C2结构域是该蛋白中与Ca（2+）和酸性磷脂结合的调节模块，现已通过与atRA共结晶获得其晶体。该晶体结构经2.0埃分辨率精修，结果显示RA在两个位置与C2结构域结合，这两个位置与先前报道的酸性磷脂的两个结合位点重合。第一个结合位点对应于Ca（2+）结合口袋，Ca（2+）离子在此介导atRA与蛋白质的相互作用，就像它们介导atRA与酸性磷脂的相互作用一样。第二个结合位点对应于位于β链3和4中的保守富含赖氨酸簇。[（3）H]-atRA结合实验结合定点诱变有力地支持了这些观察结果。野生型C2结构域每摩尔蛋白质结合2摩尔atRA，而对于C2结构域变体，该比率降至1，在这些变体中，突变影响Ca（2+）配位或富含赖氨酸簇位点的完整性。atRA与酸性磷脂结合PKC之间的竞争是调节PKC（α）活性的一种可能机制。
