Point of
VIEW
Trapping Transient Protein–Protein Interactions in Polyketide Biosynthesis Nathan A. Schnarr and Chaitan Khosla*
Departments of Chemistry, Chemical Engineering, and Biochemistry, Stanford University, Stanford, California 94305
D
espite the enormous impact of transient biomolecular interactions on chemistry and biology, reliable strategies for characterizing them remain largely elusive. Chemical cross-linking of proteins with variable binding affinities provides a means of assaying critical contact sites (1–6). For certain specialized cases, natural enzymatic activities may be exploited to serve as highly selective covalent stabilization. In this issue of ACS Chemical Biology, Burkart and co-workers describe an impressive application of this concept to fatty acid synthase components (7). This work not only will open doors to an increased understanding of thiotemplate assembly but will offer significant potential for improved biosynthetic engineering efforts. Carrier proteins (CPs) serve as primary workhorses in fatty acid, polyketide, and nonribosomal peptide biosynthesis, supplying the appropriate extender unit for each (8–10). In fatty acid and polyketide synthases, CPs collaborate with specific ketosynthases (KSs) to elongate the growing fatty acid or polyketide backbone by two carbon atoms. Ultimately, KS–CP recognition dictates the flow of intermediates as well as turnover numbers in these systems. Despite their importance, these protein– protein interactions are relatively weak. For example, the dissociation constant for the interaction between the actinorhodin KS and appropriate CPs is estimated to be in the 1–10 M range (11). Consequently, insight into the structural determinants of
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KS–CP recognition has been limited. Recent high-resolution structures from fatty acid and polyketide synthases have renewed scientific interest in modular architecture (12–14). However, because the CP position is unknown, the mechanistic basis for several steps in the overall catalytic cycle remains mysterious. Utilizing the native reactivity of both CP and KS domains, Burkart’s group has discovered a clever means of trapping KS–CP complexes for further analysis. Initially, a chemically synthesized alkylating group is enzymatically coupled to a CP via the phosphopantetheine prosthetic arm. When introduced to the partner KS domain, reversible binding promotes irreversible alkylation of the active-site cysteine residue, creating the aforementioned chemical cross-link (Figure 1, panel a). The authors clearly demonstrate that selectivity arises from protein–protein interactions and not protein–substrate specificity by examining biochemically unrelated CPs harboring the same alkylating functionality. Importantly, common and scalable synthetic methods can be used to prepare these reactive end groups, an asset too commonly overlooked. Attempts to generate a workable model for KS–CP recognition through crystallization of novel cross-links are ongoing and may finally reveal definitive roles for CP recognition elements. Clearly, much of our ability to effectively engineer biosynthetic assemblies relies on suitable reprogramming of individual enzymes. Because protein–protein interac-
A B S T R A C T Transient biomolecular interactions are essential for biological processes, but strategies for studying them have remained elusive. A paper in this issue shows how natural enzymatic activities can be exploited to examine protein– protein interactions in fatty acid synthase.
*Corresponding author,
[email protected].
Published online December 15, 2006 10.1021/cb600451d CCC: $33.50 © 2006 by American Chemical Society
VOL.1 NO.11 • ACS CHEMICAL BIOLOGY
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Figure 1. Cross-linking strategy to study fatty acid synthase. a) Schematic representation of KS– CP cross-linking via phosphopantetheine-tethered thiol traps. Transient binding of CP to KS leads to irreversible active-site alkylation. b) Theoretical application of cross-linking strategy to competitive CP binding. A panel of CPs treated with the KS of interest. A correlation between primary sequence and binding is readily extractable by comparison of bound and unbound material.
tions play a substantial role in all thiotemplate-based systems, significant attention has been focused on determining the factors governing these events. This is where a reliable cross-linking strategy may benefit researchers most. For a given KS domain, an array of similar CPs can be readily assayed for competitive binding advantage (Figure 1, panel b). The diminutive size of CPs should facilitate identification of bound material if a method of release from the partner KS is developed. The work presented by Burkart and co-workers will provide important mechanistic insights into assembly-line biosynthesis. Whether toward improving or disrupting specific biomolecular interfaces, the impact of a viable cross-linking strategy for mapping recognition elements is farreaching. This paper, together with recent structural information, may soon bring us a near-complete picture for both fatty acid and 680
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polyketide biogenesis. In all, this is another fitting example of integrative chemistry and biology.
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