Trapping the dynamic acyl carrier protein in fatty acid biosynthesis.
Trapping the dynamic acyl carrier protein in fatty acid biosynthesis.
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酰基载体蛋白（ACP）在生物合成过程中在脂肪酸合成酶（FAS）的酶域之间转运生长中的脂肪酸链。由于FAS酶作用于ACP结合的酰基，ACP必须稳定和运输不断增长的脂质链。ACP-酶相互作用的瞬时性质对获得关于脂肪酸生物合成的高分辨率结构信息施加了主要障碍，并且需要新的策略来适当地研究蛋白质-蛋白质相互作用。在这项工作中，我们描述了一个基于机制的探针，允许位点选择性共价交联的AcpP的FabA，E。coli ACP和脂肪酸3-羟酰基-ACP脱氢酶。我们报道了交联的AcpP=FabA复合物作为同源二聚体的1.9 kDa晶体结构，其中AcpP表现出两种不同的构象，可能代表ACP在作用中的快照：AcpP的4′-磷酸泛酰巯基乙胺（PPant）基团首先结合FabA的富含丝氨酸的凹槽，然后是AcpP螺旋构象变化，将AcpP和FabA锁定在适当的位置。残留物在界面的AcpP和FabA的确定和验证的解决方案NMR技术，包括化学位移扰动和RDC测量。这些不仅支持我们对晶体结构的解释，而且还提供了脂肪酸脱水过程中ACP作用的动画视图。结合分子动力学模拟，我们首次表明，FabA挤出螯合的酰基链从ACP结合口袋脱水前重新定位螺旋III。载体蛋白之间广泛的序列保守性表明，从我们的研究中收集到的机制的见解将被证明是一般的脂肪酸，聚酮化合物和非核糖体生物合成。在这里，为定义载体蛋白活性在初级和次级代谢中的动态作用奠定了基础，为深入了解在癌症、肥胖和传染病治疗中发挥重要作用的途径提供了依据。
Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis. Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain. The transient nature of ACP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of a mechanism-based probe that allows site-selective covalent crosslinking of AcpP to FabA, the E. coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase. We report the 1.9 Å crystal structure of the crosslinked AcpP=FabA complex as a homo-dimer, in which AcpP exhibits two different conformations likely representing snapshots of ACP in action: the 4′-phosphopantetheine (PPant) group of AcpP first binds an arginine-rich groove of FabA, followed by an AcpP helical conformational change that locks the AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution NMR techniques, including chemical shift perturbations and RDC measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. Combined with molecular dynamics simulations, we show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies will prove general for fatty acid, polyketide and non-ribosomal biosyntheses. Here the foundation is laid for defining the dynamic action of carrier protein activity in primary and secondary metabolism, providing insight into pathways that can play major roles in the treatment of cancer, obesity and infectious disease.