Department of Chemistry, Syracuse University, Syracuse, New York 13244.
Department of Biomedical and Chemical Engineering, Syracuse University, Syracuse, New York 13244.
J Biol Chem. 2019 Sep 27;294(39):14166-14174. doi: 10.1074/jbc.AC119.009749. Epub 2019 Aug 14.
Integral membrane proteins represent a large and diverse portion of the proteome and are often recalcitrant to purification, impeding studies essential for understanding protein structure and function. By combining co-evolutionary constraints and computational modeling with biochemical validation through site-directed mutagenesis and enzyme activity assays, we demonstrate here a synergistic approach to structurally model purification-resistant topologically complex integral membrane proteins. We report the first structural model of a eukaryotic membrane-bound -acyltransferase (MBOAT), ghrelin acyltransferase (GOAT), which modifies the metabolism-regulating hormone ghrelin. Our structure, generated in the absence of any experimental structural data, revealed an unanticipated strategy for transmembrane protein acylation with catalysis occurring in an internal channel connecting the endoplasmic reticulum lumen and cytoplasm. This finding validated the power of our approach to generate predictive structural models for other experimentally challenging integral membrane proteins. Our results illuminate novel aspects of membrane protein function and represent key steps for advancing structure-guided inhibitor design to target therapeutically important but experimentally intractable membrane proteins.
整合膜蛋白是蛋白质组中一个庞大而多样的部分，通常难以纯化，这阻碍了对蛋白质结构和功能的理解至关重要的研究。通过将共进化约束和计算建模与通过定点突变和酶活性测定进行的生化验证相结合，我们在这里展示了一种协同方法来对具有拓扑复杂性的纯化抗性整合膜蛋白进行结构建模。我们报告了第一个真核膜结合酰基转移酶（MBOAT），即胃饥饿素酰基转移酶（GOAT）的结构模型，该酶修饰代谢调节激素胃饥饿素。我们的结构是在没有任何实验结构数据的情况下生成的，揭示了一种用于跨膜蛋白酰化的出乎意料的策略，该策略发生在连接内质网腔和细胞质的内部通道中。这一发现验证了我们生成其他具有实验挑战性的整合膜蛋白的预测结构模型的方法的有效性。我们的结果阐明了膜蛋白功能的新方面，并代表了推进基于结构的抑制剂设计以靶向治疗上重要但实验上难以处理的膜蛋白的关键步骤。
