Voltage-gated potassium channels (Kv) are gated by the movement of the transmembrane voltage sensor, which is coupled, through the helical S4–S5 linker, to the potassium pore. We determined the single-particle cryo-EM structure of mammalian Kv10.1 or Eag1, bound to the channel inhibitor calmodulin, at 3.78Å resolution. Unlike previous Kv structures, the S4–S5 linker of Eag1 is a 5-residue loop and the transmembrane segments are not domain swapped, suggesting an alternative mechanism of voltage-dependent gating. Additionally, the structure and position of the S4–S5 linker allows calmodulin to bind to the intracellular domains and close the potassium pore independent of voltage sensor position. The structure reveals an alternative gating mechanism for Kv channels and provides a template to further understand the gating properties of Eag1 and related channels.

Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two-carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We employed electron cryo-microscopy to visualize a full-length module and determine sub-nanometer resolution 3D reconstructions that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intra-module carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time the structural basis for both intra-module and inter-module substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents1. The architecture of a full-length PKS module from the pikromycin pathway creates a reaction chamber for the intra-module acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) active sites (see accompanying paper by Dutta et al.). Here we determined electron cryo-microscopy (cryo-EM) structures of a full-length PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC/FT-ICR MS). The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of KS, after extension to the β-keto-intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential binding to catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the KR domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

SUMMARY Modular type I polyketide synthases (PKSs) are versatile biosynthetic systems that initiate, successively elongate and modify acyl chains. Intermediate transfer between modules is mediated via docking domains, which are attractive targets for PKS pathway engineering to produce novel small molecules. We identified a Class 2 docking domain in cyanobacterial PKSs and determined crystal structures for two docking domain pairs, revealing a novel docking strategy for promoting intermediate transfer. The selectivity of Class 2 docking interactions, demonstrated in binding and biochemical assays, could be altered by mutagenesis. We determined the ideal fusion location for exchanging Class 1 and Class 2 docking domains and demonstrated effective polyketide chain transfer in heterologous modules. Thus, Class 2 docking domains are new tools for rational bioengineering of a broad range of PKSs containing either Class 1 or 2 docking domains.