Kelsey Jessica S, Geczy Tamas, Lewin Nancy E, Kedei Noemi, Hill Colin S, Selezneva Julia S, Valle Christopher J, Woo Wonhee, Gorshkova Inna, Blumberg Peter M
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute Building 37, Room 4048, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255, U.S.A.
Biomedical Engineering and Physical Science Share Resource Program, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, U.S.A.
Chembiochem. 2014 May 26;15(8):1131-1144. doi: 10.1002/cbic.201400041. Epub 2014 Apr 28.
The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters, has emerged as a promising therapeutic target for cancer and other indications. Potential target selectivity is markedly enhanced both because binding reflects ternary complex formation between the ligand, C1 domain, and phospholipid, and because binding drives membrane insertion of the C1 domain, permitting aspects of the C1 domain surface outside the binding site, per se, to influence binding energetics. Here, focusing on charged residues identified in atypical C1 domains which contribute to their loss of ligand binding activity, we showed that increasing charge along the rim of the binding cleft of the protein kinase C δ C1 b domain raises the requirement for anionic phospholipids. Correspondingly, it shifts the selectivity of C1 domain translocation to the plasma membrane, which is more negatively charged than internal membranes. This change in localization is most pronounced in the case of more hydrophilic ligands, which provide weaker membrane stabilization than do the more hydrophobic ligands and thus contributes an element to the structure-activity relations for C1 domain ligands. Coexpressing pairs of C1-containing constructs with differing charges each expressing a distinct fluorescent tag provided a powerful tool to demonstrate the effect of increasing charge in the C1 domain.
C1结构域是蛋白激酶C上识别亲脂性第二信使二酰基甘油及其超效类似物佛波酯的基序，已成为治疗癌症和其他适应症的一个有前景的治疗靶点。潜在的靶点选择性显著增强，这既是因为结合反映了配体、C1结构域和磷脂之间的三元复合物形成，也是因为结合驱动C1结构域插入膜中，使得结合位点之外的C1结构域表面部分能够影响结合能。在此，我们聚焦于非典型C1结构域中导致其丧失配体结合活性的带电荷残基，结果表明，沿着蛋白激酶C δ C1 b结构域结合裂隙边缘增加电荷会提高对阴离子磷脂的需求。相应地，它会使C1结构域向质膜转位的选择性发生改变，质膜比内膜带更多负电荷。在更亲水性配体的情况下，这种定位变化最为明显，亲水性配体比疏水性配体提供的膜稳定性更弱，因此这为C1结构域配体的构效关系提供了一个要素。共表达带有不同电荷且各自表达不同荧光标签的一对含C1结构域的构建体，为证明增加C1结构域电荷的效应提供了一个有力工具。
