Ochoa Wendy F, Corbalán-Garcia Senena, Eritja Ramon, Rodríguez-Alfaro José A, Gómez-Fernández Juan C, Fita Ignacio, Verdaguer Nuria
Instituto de Biología Molecular de Barcelona (CSIC), Jordi Girona Salgado 18-26, E-08034 Barcelona, Spain.
J Mol Biol. 2002 Jul 5;320(2):277-91. doi: 10.1016/S0022-2836(02)00464-3.
The C2 domain of protein kinase Calpha (PKCalpha) corresponds to the regulatory sequence motif, found in a large variety of membrane trafficking and signal transduction proteins, that mediates the recruitment of proteins by phospholipid membranes. In the PKCalpha isoenzyme, the Ca2+-dependent binding to membranes is highly specific to 1,2-sn-phosphatidyl-l-serine. Intrinsic Ca2+ binding tends to be of low affinity and non-cooperative, while phospholipid membranes enhance the overall affinity of Ca2+ and convert it into cooperative binding. The crystal structure of a ternary complex of the PKCalpha-C2 domain showed the binding of two calcium ions and of one 1,2-dicaproyl-sn-phosphatidyl-l-serine (DCPS) molecule that was coordinated directly to one of the calcium ions. The structures of the C2 domain of PKCalpha crystallised in the presence of Ca2+ with either 1,2-diacetyl-sn-phosphatidyl-l-serine (DAPS) or 1,2-dicaproyl-sn-phosphatidic acid (DCPA) have now been determined and refined at 1.9 A and at 2.0 A, respectively. DAPS, a phospholipid with short hydrocarbon chains, was expected to facilitate the accommodation of the phospholipid ligand inside the Ca2+-binding pocket. DCPA, with a phosphatidic acid (PA) head group, was used to investigate the preference for phospholipids with phosphatidyl-l-serine (PS) head groups. The two structures determined show the presence of an additional binding site for anionic phospholipids in the vicinity of the conserved lysine-rich cluster. Site-directed mutagenesis, on the lysine residues from this cluster that interact directly with the phospholipid, revealed a substantial decrease in C2 domain binding to vesicles when concentrations of either PS or PA were increased in the absence of Ca2+. In the complex of the C2 domain with DAPS a third Ca2+, which binds an extra phosphate group, was identified in the calcium-binding regions (CBRs). The interplay between calcium ions and phosphate groups or phospholipid molecules in the C2 domain of PKCalpha is supported by the specificity and spatial organisation of the binding sites in the domain and by the variable occupancies of ligands found in the different crystal structures. Implications for PKCalpha activity of these structural results, in particular at the level of the binding affinity of the C2 domain to membranes, are discussed.
蛋白激酶Cα（PKCα）的C2结构域对应于一种调节序列基序，该基序存在于多种膜运输和信号转导蛋白中，介导磷脂膜对蛋白的募集。在PKCα同工酶中，Ca2+依赖性膜结合对1,2-二酰基-sn-磷脂酰-L-丝氨酸具有高度特异性。内在Ca2+结合往往亲和力较低且不具有协同性，而磷脂膜可增强Ca2+的整体亲和力并将其转化为协同结合。PKCα-C2结构域三元复合物的晶体结构显示了两个钙离子和一个1,2-二己酰基-sn-磷脂酰-L-丝氨酸（DCPS）分子的结合，该DCPS分子直接与其中一个钙离子配位。现已分别在1.9 Å和2.0 Å分辨率下测定并优化了在Ca2+存在下与1,2-二乙酰基-sn-磷脂酰-L-丝氨酸（DAPS）或1,2-二己酰基-sn-磷脂酸（DCPA）结晶的PKCα C2结构域的结构。DAPS是一种具有短烃链的磷脂，预计它有助于磷脂配体在Ca2+结合口袋内的容纳。DCPA带有磷脂酸（PA）头部基团，用于研究对具有磷脂酰-L-丝氨酸（PS）头部基团的磷脂的偏好。所测定的这两种结构显示在保守的富含赖氨酸簇附近存在一个阴离子磷脂的额外结合位点。对该簇中直接与磷脂相互作用的赖氨酸残基进行定点诱变发现，在没有Ca2+的情况下，当PS或PA浓度增加时，C2结构域与囊泡的结合显著减少。在C2结构域与DAPS的复合物中，在钙结合区域（CBRs）中鉴定出第三个结合额外磷酸基团的Ca2+。PKCα C2结构域中钙离子与磷酸基团或磷脂分子之间的相互作用得到该结构域中结合位点的特异性和空间组织以及不同晶体结构中发现配体占据情况的支持。讨论了这些结构结果对PKCα活性的影响，特别是在C2结构域对膜的结合亲和力水平上的影响。
