Journal:Acta Cryst D:S2059798322004612

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural Visualization of Transient Interactions Between the cis-acting Acyltransferase and Acyl Carrier Protein of Salinomycin Modular Polyketide Synthase Y. Feng, F. Zhang, S. Huang, Z. Deng, L. Bai and J. Zheng Molecular Tour Polyketides, such as erythromycin, ivermectin, and rifamycin, are a large group of clinically important natural products. Although structurally diverse, they are synthesized by polyketide synthases (PKSs) from relatively simple short-chain carboxylic acids activated by coenzyme A (CoA). The assembly of a polyketide chain is similar to that of a fatty acid chain. Usually, a monocarboxylic acid is utilized as a starter unit and condensed with dicarboxylic acid extender units iteratively until the polyketide chain reaches an appropriate length. The selection of various starter and extender units by an acyltransferase (AT) is the first step of polyketide chain extension. An AT loads an acyl unit onto the phosphopantetheine thiol of an acyl carrier protein (ACP) domain, where it is used in the decarboxylative Claisen condensation catalyzed by a ketosynthase (KS) to elongate the polyketide chain. Modular PKSs (mPKSs) containing covalently fused domains for catalytic cycles are among the most versatile megaenzymes, of which each module is responsible for a single round of polyketide chain elongation. Canonical mPKSs contain a cis-acting AT domain (cis-AT) in every module. However, mPKSs that have modules without embedded AT domains are also discovered and utilize external trans-acting ATs (trans-ATs), which are standalone enzymes, to provide acyl units for polyketide chain elongation. The apparent architectural modularity encourages the construction of hybrid mPKSs to generate unnatural polyketides. The cis-AT domains control the acyl unit incorporated into every elongation step and therefore are attractive engineering targets. This highlights the need for more careful consideration of proper AT-ACP interactions in engineering mPKSs. The inherently transient and weak nature is the key challenge in understanding protein-protein interactions between AT and ACP. The weak mutual binding affinity hampers the structural determination of a AT-ACP complex for directly visualization of protein-protein interactions. We reported the crystal structure of a cis-AT-ACP complex (PDB ID: 7vrs) from the 9th extension module of a salinomycin mPKS. SalAT9 is organized into a large α,β-hydrolase subdomain (residues 97−224 and 299−418) and a small ferredoxin-like subdomain (residues 228−294). The N-terminal KS-to-AT linker (residues 1-92) forms a three-stranded antiparallel β-sheet and three α-helices, packing against the AT domain. The transient cis-AT-ACP complex was obtained by using 1,4-bis(maleimido)butane (BMB) as a crosslinking agent. The BMB maleimide groups covalently connect the AT and ACP. The ACP-binding mode in the cis-AT-ACP complex structure is strikingly different from those of the previously reported trans-AT-ACP complex structures. The ACP primarily contacts the large subdomain of the AT in structure of the cis-AT-ACP complex, whereas the ACP primarily contacts the small domain of the AT in the trans-AT-ACP complex structures. The complex structure provides detailed mechanistic insights into the protein-protein interactions between AT and ACP domains of cis-AT mPKSs and could potentially help optimize chimeric cis-AT mPKSs for unnatural polyketides. SalAT9M large hydrolase subdomain (in dark turquoise) functions as the major binding platform for SalACP9 (in burlywood): The SalAT9M-ACP9 complex structure, transparent surface representation. The αII helix of SalACP9 packs against the helix α8 (residues 378-388) of the large hydrolase subdomain with an angle of 26°. The short helix αIII’ of the loop II of SalACP9 packs against the loop C of the small subdomain of SalAT9M. In the crystallographic asymmetric unit, a SalAT9M-ACP9 complex and a standalone SalAT9M molecule were observed. SalACP9 binding induces SalAT9M to a more open state compared to the standalone SalAT9M’. Animation of previous scene. The KS-to-AT linkers have been shown to functional as a structural element stabilizing the ATs that is expressed as standalone domains. The absence or incompleteness of KS-to-AT linkers in AT constructs may result in insoluble or inactive proteins. The complex structure shows that the loop IV (residues 80-86) of the KS-to-AT linker makes contacts with the N-terminal residues of loop I of SalACP9. PDB references: The complex of Acyltransferase and Acyl Carrier Protein Domains from module 9 of Salinomycin Polyketide Synthase, 7vrs; Acyltransferase from the 9th Module of Salinomycin Polyketide Synthase, 7vt1. References

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