S143: Architecture and dynamics of mega-synthases involved in primary and secondary metabolism

The carrier protein (CP) transports the growing intermediates between enzyme domains of fatty acid synthase (FAS), polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) in primary and secondary metabolism. Because these mega-synthases operate upon CP-bound substrates, CP must stabilize and transport the growing chain. The transient nature of CP-enzyme interactions imposes a major obstacle to gaining high-resolution structural information about biosynthesis directed by the mega-synthases, and a new strategy is required to properly study protein-protein interactions. In this work, we describe the application of mechanism-based probes that allow site-selective covalent crosslinking of CP to downstream enzymes. We report the 1.9 Å crystal structure of a crosslinked complex of an acyl carrier protein (ACP) with its enzyme partner, in which ACP exhibits two different conformations representing snapshots of ACP in action: the 4’-phosphopantetheine (PPant) group of ACP first binds an arginine-rich groove of its partner enzyme, followed by an ACP helical conformational change that locks the ACP and its partner enzyme in place. Residues at the interface of ACP and its partner are further identified and validated by solution-phase NMR techniques, including HSQC perturbation and RDC measurements. Combined with molecular dynamic simulation, we show for the first time that the mega-synthase extrudes the sequestered substrate from the ACP binding pocket by repositioning helix III. 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.