Corbalán-Garcia Senena, Sánchez-Carrillo Susana, García-García Josefa, Gómez-Fernández Juan C
Department de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo. 4021, E-30100 Murcia, Spain.
Biochemistry. 2003 Oct 14;42(40):11661-8. doi: 10.1021/bi034850d.
PKCepsilon is a member of the group of novel PKCs that contain a C2 domain located in their N-terminal region. On the basis of recent structural studies, a series of mutants were prepared to increase our knowledge of the mechanism of the phospholipid binding site of this domain. The results revealed that this domain preferentially binds to phosphatidic acid- and phosphatidylserine-containing vesicles. Although the increase in affinity was linear in the case of phosphatidic acid, it became exponential when the vesicles contained increasing concentrations of phosphatidylserine. Site-directed mutagenesis studies showed that residues W23, R26, and R32 located in loop 1 and I89 and Y91 located in loop 3 are of critical importance when the binding is performed with phosphatidic acid-containing vesicles. Furthermore, when the same mutants were assayed with phosphatidylserine-containing vesicles, no binding was observed in any case, reflecting the smaller affinity of the C2 domain for phosphatidylserine-containing vesicles. A study of the ionic nature of the membrane interaction suggested that it is mainly driven by electrostatic interactions that are disrupted by very low salt concentrations. Differential scanning calorimetry experiments performed to ascertain whether this interaction affected the transition phase of the phosphatidic acid demonstrated that increasing concentrations of the protein lead to changes in the transition, with more than one peak appearing at lower temperatures, which suggests a weak interaction focused on the polar headgroup of the phospholids. In conclusion, a different membrane-binding mode from those previously described in other C2 domains has been found and is seemingly based on electrostatic, interfacial, and hydrophobic interactions without the participation of Ca(2+) ions.
蛋白激酶Cε（PKCε）是新型蛋白激酶C家族的成员之一，其N端区域含有一个C2结构域。基于近期的结构研究，制备了一系列突变体，以增进我们对该结构域磷脂结合位点机制的了解。结果表明，该结构域优先结合含磷脂酸和磷脂酰丝氨酸的囊泡。虽然在磷脂酸的情况下亲和力的增加呈线性，但当囊泡中磷脂酰丝氨酸浓度增加时，亲和力的增加呈指数关系。定点诱变研究表明，当与含磷脂酸的囊泡结合时，位于环1中的W23、R26和R32残基以及位于环3中的I89和Y91残基至关重要。此外，当用含磷脂酰丝氨酸的囊泡对相同的突变体进行检测时，在任何情况下都未观察到结合，这反映了C2结构域对含磷脂酰丝氨酸囊泡的亲和力较小。对膜相互作用的离子性质的研究表明，它主要由静电相互作用驱动，而极低的盐浓度会破坏这种相互作用。为确定这种相互作用是否影响磷脂酸的转变相而进行的差示扫描量热实验表明，蛋白质浓度的增加会导致转变发生变化，在较低温度下出现多个峰，这表明相互作用较弱，集中在磷脂的极性头部基团上。总之，发现了一种与其他C2结构域先前描述的不同的膜结合模式，这种模式似乎基于静电、界面和疏水相互作用，而不涉及钙离子的参与。
