Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

Abstract

Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

“…During the continuously maturation of the intermediate, ACP must form specific protein-protein interactions, and the tethered intermediate should be translocation from the hydrophobic core of ACP into the active site of the partners. The ACP-mediated assembly pattern in the natural product biosynthesis pathway offers a safe, efficient, and economical program to facilitate the direct production of the final compounds [22,30,32]. Combined with our present investigations, the AHBA-tethered MmcB triggered the biosynthesis of the early-stage of mitomycin C. In this reaction, MmcB specifically interacted with the partners, such as MitE, MitF, and others, to afford the mitosane skeleton.…”

“…Remarkably, the ACP-dependent tailoring enzymes are involving in the biosynthesis of a large number of important chemical molecules, from primary metabolites to structurally complex natural products [18,29,30]. The ACP is a relatively small protein with a dynamic and monomeric helical bundle architecture, which shuttles the nascent cargo to the partner enzymes for decoration through the biosynthetic machinery.…”

“…The ACP is a relatively small protein with a dynamic and monomeric helical bundle architecture, which shuttles the nascent cargo to the partner enzymes for decoration through the biosynthetic machinery. In the biosynthesis of natural product, the nascent intermediate is covalently tethered to the holo-ACP via thioester linkage to a post-translationally substituted phosphopantetheine (PPant) arm, which is activated by PPTase from the apo-ACP [29,30]. Moreover, the ACP protects the growing nascent intermediates from non-selective modification or degradation in the cytosol by sequestering them within a central hydrophobic core [31].…”

Protein–protein interaction based substrate control in the E. coli octanoic acid transferase, LipB

“…The resulting docking poses were organized based on RMSD from the previously published model of the C8-AcpP • LipB docked complex 20 (Figure S10). The methodology used was the same as we previously developed to model six ACP-partner protein interfaces from E. coli FAB and benchmark them with established crystal crosslinked structures 23 .…”

“…This data was then used to guide high-resolution in silico docking to identify the surface features responsible for the experimentally identified binding differences. We have shown that the implementation of NMR titration experiments to guide docking algorithms can accurately and reproducibly deduce PPI poses in ACP-dependent pathways 23 .…”