Wang S, Kazanietz M G, Blumberg P M, Marquez V E, Milne G W
Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
J Med Chem. 1996 Jun 21;39(13):2541-53. doi: 10.1021/jm950403n.
The protein kinase C (PKC) binding site used by PKC activators such as phorbol esters and diacylglycerols (DAGs) has been characterized by means of molecular modeling and site-directed mutagenesis studies. Based upon a NMR-determined solution structure of the second cysteinerich domain of PKC alpha, molecular modeling was used to study the structures of the complexes formed between the PKC receptor and a number of PKC ligands, phorbol esters, and DAGs. Site-directed mutagenesis studies identified a number of residues important to the binding of phorbol esters to PKC. Analysis of the molecular modeling and mutagenesis results allows the development of a binding model for PKC ligands for which the precise binding nature is defined. The calculated hydrogen bond energies between the protein and various ligands in this binding model are consistent with their measured binding affinities. The binding site for phorbol esters and DAGs is located in a highly conserved, hydrophobic loop region formed by residues 6-12 and 20-27. For the binding elements in phorbol esters, the oxygen at C20 contributes most to the overall binding energy, and that at C3 plays a significant role. The oxygen atom at C12 is not directly involved in the interaction between phorbol esters and PKC. Our results also suggest that the oxygens at C9 and C13 are involved in PKC binding, while the oxygen at C4 is of minimal significance. These results are consistent with known structure-activity relationships in the phorbol ester family of compounds. Comparisons with the X-ray structure showed that although the X-ray data support the results for oxygens at C3, C12, and C20 of phorbol esters, they suggest different roles for oxygens at C4, C9, and C13. Several factors which may contribute to these discrepancies are discussed.
蛋白激酶C（PKC）激活剂（如佛波酯和二酰基甘油（DAGs））所使用的PKC结合位点已通过分子建模和定点诱变研究得以表征。基于PKCα第二个富含半胱氨酸结构域的核磁共振确定的溶液结构，利用分子建模研究了PKC受体与多种PKC配体、佛波酯和DAGs形成的复合物的结构。定点诱变研究确定了一些对佛波酯与PKC结合很重要的残基。对分子建模和诱变结果的分析有助于建立一个PKC配体的结合模型，该模型明确了精确的结合性质。此结合模型中蛋白质与各种配体之间计算出的氢键能与其测得的结合亲和力一致。佛波酯和DAGs的结合位点位于由残基6 - 12和20 - 27形成的高度保守的疏水环区域。对于佛波酯中的结合元件，C20位的氧对整体结合能贡献最大，C3位的氧起重要作用。C12位的氧不直接参与佛波酯与PKC之间的相互作用。我们的结果还表明，C9和C13位的氧参与PKC结合，而C4位的氧意义最小。这些结果与佛波酯类化合物中已知的构效关系一致。与X射线结构的比较表明，尽管X射线数据支持佛波酯C3、C12和C20位氧的结果，但它们表明C4、C9和C13位氧的作用不同。讨论了可能导致这些差异的几个因素。
