Trapping the dynamic acyl carrier protein in fatty acid biosynthesis

Abstract

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzyme domains of fatty acid synthase (FAS) during biosynthesis.1 Because FAS enzymes operate upon ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain.2 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.

CRISPR-Cas9 conformational activation as elucidated from enhanced molecular simulations

“…Along this pathway, 24 equally distributed frames-including the crystallographic states-were subjected to conventional MD and GaMD simulations for a total of ∼15 μs with the aim of rigorously characterizing the conformational dynamics of the complex over a long timescale, made possible through the application of enhanced sampling (SI Appendix) (15,16,19). Intermolecular FRET distances have been used to characterize the conformational states of Cas9 during the dynamics ( Fig.…”

“…We perform accelerated MD simulations, which have been demonstrated to provide routine access to events occurring on and-in some cases-beyond the millisecond timescale (15,16), complemented by the application of biasing methods (17), enabling the investigation of intermediate states for which structural characterization is absent. We use a Gaussian-accelerated MD (GaMD) (18) method that accelerates biomolecular conformational transitions between low-energy states by smoothing the potential energy surface with a harmonic boost potential that follows a Gaussian distribution.…”

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

“…7C), which, in combination with the inherent flexibility of the carrier arm, would allow the carrier arm to be accommodated within active sites of different structure. A paucity of carrier arm interactions with the partner enzyme active site has also been observed for phosphopantetheine-containing carrier proteins, suggesting that this strategy is a general feature of carrier protein interactions (6,(29)(30)(31).…”

“…7B). The conformational change undergone by the SoxYZ carrier arm on complex formation with SoxB is reminiscent of the "switchblade" mechanism used by acyl carrier proteins in which the nonpolar substrate molecule bound to the phosphopantetheine arm is protected within a hydrophobic pocket on the side of the carrier protein, but swung fully out of the pocket to insert into the partner enzyme on complex formation (6).…”

“…In other cases, the carrier protein and its partner enzymes are separate entities that form transient complexes to effect catalysis. Examples include the acyl carrier proteins interacting with enzymes involved in type II fatty acid synthesis and polyketide synthesis (5,6). In reality, these two types of carrier protein pathways are mechanistically similar because the carrier protein domain in the permanent complexes is normally itself flexibly tethered to the rest of the complex, allowing significant freedom of movement between partner enzyme domains (1,2).…”