Chasing Acyl-Carrier-Protein Through a Catalytic Cycle of Lipid A Production

Acyl-carrier-protein (ACP) represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon-chains in vivo. Notably, type II ACPs serve as a crucial interaction hub within primary cellular metabolism(1) by communicating transiently between partner enzymes of the numerous biosynthetic pathways(2,3). However, the highly transient nature of such interactions and the inherent conformational mobility of ACP(2) have stymied previous attempts to structurally visualize ACP tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but are also of therapeutic value. For example, ACP is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides (LPS) in Gram-negative microorganisms, which is required for their growth and survival(4,5) and is an activator of the mammalian host's immune system(6,7), thus emerging as an important therapeutic target(8-10). During lipid A synthesis (Raetz Pathway), ACP shuttles acyl-intermediates linked to its prosthetic 4′-phosphopantetheine group (4′-PPT)(2) among four acyltransferases, including LpxD(11). Here we report the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release.