Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking

Abstract

Probing and interrogating protein interactions that involve acyl carrier proteins (ACP's) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACP's, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.

A Protein Interaction Map of the Kalimantacin Biosynthesis Assembly Line

“…The interaction of the KS1 fragment with BatJ supports this hypothesis, however, docking of BatJ to this region would be expected to occur in each module. Recently, different studies have shown that trans -AT domains can directly transfer their acyl group to the ACP domain, even in the absence of a KS domain or post-KS linker region and suggestions rise that the trans -AT domains do not even dock to the mega enzyme cluster (Aron et al, 2007; Musiol et al, 2011; Wong et al, 2011; Ye and Williams, 2014). This could explain the low amount of interactions seen for BatJ.…”

Late‐Stage Direct o‐Alkenylation of Phenols by PdII‐Catalyzed C−H Functionalization

“…In addition, styrene and its derivatives containing electron‐donating and ‐withdrawing substituents reacted well with phenol to give ( E )‐2‐styrylphenols 3 k , l , and m in reasonable yields. It is also noteworthy that a benzophenone connected with an acrylic ester could be successfully reacted with phenols ( 3 e ), in which benzophenone has been known to serve as a photo‐cross‐linking group for activity‐based profiling probes (ABPP) and to improve the biological activity of target drug molecules …”

“…It is also noteworthy that ab enzophenone connected with an acrylice ster could be successfully reactedw ith phenols( 3e), in which benzophenone has been known to serve as ap hotocross-linking group fora ctivity-based profiling probes (ABPP) and to improve the biological activity of target drug molecules. [11] It is important to highlight that the phenolic OÀHa nd C=C groups in the olefination product couldp rovide flexibility for furthers tructurald iversification (Scheme2a). To further show the widea pplicability of this method, 2a was converted into a benzofuran derivative 4a and ac oumarin derivative 4b in good yields.…”

Harnessing natural product assembly lines: structure, promiscuity, and engineering

“…For example, benzophenone has been site-specifically installed into carrier proteins via amber suppression technology and has been used to probe KS-ACP interactions by photocrosslinking and quantification of the crosslinked species ( Figure 3b ) [149]. In combination with surface scanning mutagenesis, this strategy has been used to map an ACP:KS binding interface [151]. Additionally, unnatural extender units modified with ‘click chemistry’ handles have been used to probe protein-protein interactions.…”