Decoding allosteric regulation by the acyl carrier protein

Abstract

Significance Acyl carrier proteins (ACPs) are involved in primary and secondary metabolic pathways, including the ubiquitous fatty acid biosynthesis, required for all domains of life. This single protein must deliver pathway intermediates to the appropriate enzyme, distinguishing between a myriad of possible intermediate-enzyme combinations. The intermediate is delivered to the active site of enzymes through a large conformational change termed “chain flipping.” Whether chain flipping is a stochastic or regulated process has remained a mystery. This study provides evidence for an allosteric regulatory mechanism—demonstrating that substrates sequestered within the interior side of the four-helical ACP bundle confer structural changes to the exterior which are recognized by enzymes via protein–protein interactions—presenting a unique paradigm for understanding these biosynthetic pathways. 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, Microbiol. Rev. 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., Chem. Biol. 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, ChemBioChem 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.