Sztain Terra, Bartholow Thomas G, Lee D John, Casalino Lorenzo, Mitchell Andrew, Young Megan A, Wang Jianing, McCammon J Andrew, Burkart Michael D
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0358.
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093-0358;
Proc Natl Acad Sci U S A. 2021 Apr 20;118(16). doi: 10.1073/pnas.2025597118.
Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, 57, 522-542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., 17, 776-785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein-protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, 16, 528-547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein-protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.
多步代谢途径中的酶利用一系列调节机制来维持微妙的体内平衡[K. 马格努森、S. 雅科夫斯基、C. O. 罗克、J. E. 克罗南， Jr， 57, 522 - 542 (1993)]。载体蛋白在代谢途径中在合适的酶之间穿梭底物方面尤其起着至关重要的作用。尽管有过相关假说[E. 普沃斯科恩等人， 17, 776 - 785 (2010)]，但对于任何依赖酰基载体蛋白(ACP)的途径，底物传递的变构调节此前从未得到证实。由于蛋白质 - 蛋白质相互作用的瞬态和动态性质、底物的巨大多样性以及底物不稳定性[K. 芬泽尔、D. J. 李、M. D. 伯克哈特， 16, 528 - 547 (2015)]，研究这些机制一直具有挑战性。在这里，我们使用溶液核磁共振光谱和分子动力学来阐明依赖脂肪酸链长度的变构作用，从而证明了ACP与伙伴酶之间独特的通讯机制。我们证明，伙伴酶可以通过蛋白质 - 蛋白质相互作用对链长度进行变构区分，因为底物隔离的结构特征从ACP四螺旋束内部传递到了蛋白质表面，而无需随机的链翻转。这些结果揭示了ACP货物通讯的细节，可为设计依赖载体蛋白的途径以生产特定的所需产物奠定基础。
