Volume 20, Issue 11, Pages 1340-1351 (November 2013) Cyanobacterial Polyketide Synthase Docking Domains: A Tool for Engineering Natural Product Biosynthesis  Jonathan R. Whicher, Sarah S. Smaga, Douglas A. Hansen, William C. Brown, William H. Gerwick, David H. Sherman, Janet L. Smith  Chemistry & Biology  Volume 20, Issue 11, Pages 1340-1351 (November 2013) DOI: 10.1016/j.chembiol.2013.09.015 Copyright © 2013 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2013 20, 1340-1351DOI: (10. 1016/j. chembiol. 2013 Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Curacin Docking Domains (A) PKS portion of the curacin A (Cur) pathway from Moorea producens (Chang et al., 2004). Matched docking domain pairs used for the binding experiments are indicated with the same color. Crystallized docking domain pairs are indicated by red boxes. (B) Sequence alignment of Cur ACPdds (top, orange) and Cur ddKSs (bottom, cyan) used for the crystallization experiments extracted from a sequence alignment of 23 cyano- and myxobacterial docking domain pairs (Figure S1). Matched docking domain pairs are indicated by the same color. Residues are colored by type (hydrophobic-yellow, polar-green, acidic-red, basic-blue, and P- or G-purple) and conservation (darker colors indicate higher conservation). Secondary structure elements are annotated above the sequence alignments (rectangles are helices). Purple stars indicate amino acids involved in the extended interface of the class 2 dock, red circles indicate those involved in selectivity-promoting electrostatic interactions, and the a and d positions of the coiled coil heptad repeats are labeled. See also Figure S1. Chemistry & Biology 2013 20, 1340-1351DOI: (10.1016/j.chembiol.2013.09.015) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Class 2 Docking Domain Structures (A) GH dock. In the upper stereo diagram, monomers are shown with different shades of orange (ACPdd) or cyan (ddKS). The GlySer linker (green) connects the upstream ACPdd C terminus and the downstream ddKS N terminus. Connections to the ACP and KS domains are shown with thick lines. Close-up stereoviews are shown of the class 2-specific ACPddG α1 interface with ddHKS αA and αB (purple box) and the common ACPddG α2 interface with ddHKS αB coiled-coil (red box). Hydrophobic contacts in the docking interface are shown as sticks with orange C for the ACPdd, cyan C for the ddKS, red O, and blue N. (B) KL dock. Coloring and labeling are as in (A). Zoomed-in stereoviews of the boxed regions of the upper stereo diagram are highlighted below. Electrostatic interactions that may promote selectivity are labeled, and hydrogen bonds are indicated with red dashes (detail in Figures S2E and S2F). See also Figure S2. Chemistry & Biology 2013 20, 1340-1351DOI: (10.1016/j.chembiol.2013.09.015) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 Superposition of Class 2 ddKS and ACPdd (A) Superposition of class 2 ddKS. The CurL ddKS from KL dock structure (cyan) was superimposed with the CurL ddKS (yellow) from the CurL dd-KS-AT structure (root-mean-square deviation [rmsd] = 1.3 Å) and with the CurG ddKS (magenta) from the GH dock structure (rmsd = 1.0 Å). Superposition of Fo-Fc simulated annealing (SA) density contoured at 3σ for the ddKS loops from each of the three structures are shown below the superposition. (B) Superposition of the class 2 ACPdd. The ACPdd from GH dock structure (yellow) and KL dock (orange) were superimposed (rmsd = 0.564 Å). Fo-Fc SA density contoured at 3σ for the ACPdd loops from the GH and KL dock structures are shown below the superposition. Chemistry & Biology 2013 20, 1340-1351DOI: (10.1016/j.chembiol.2013.09.015) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 Comparison of Class 2 and Class 1 Docking Domains The class 2 GH dock (top left) and KL dock (top right) (orange ACPdd and cyan ddKS) were modeled onto the CurL dd-KS-AT (blue KS, green AT, red KS-AT linker domain, KS active site cysteine in yellow spheres). The class 1 dimerization helices (orange) and ACPdd (orange)/ddKS (cyan) complex from the DEBS module 2–3 interface (Broadhurst et al., 2003) was modeled onto the KS-AT di-domain from DEBS module 5 (Tang et al., 2006). In the class 2 GH dock (top left), the ACP is directed toward the downstream KS, whereas the KS and ACP are much further apart in the class 1 dock. Flexibility in the class 2 dock, as seen in the KL dock (top right) allows the upstream ACP to interact with catalytic domains of the upstream module. Chemistry & Biology 2013 20, 1340-1351DOI: (10.1016/j.chembiol.2013.09.015) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 5 PikAIII/PikAIV Assay of Docking Effectiveness (A) Schematic of the two-module throughput reaction. The 10-dml and nbl macrocycles are formed by the PikAIV TE following processing of the pentaketide substrate by PikAIII and PikAIII/PikAIV, respectively. (B) Assay results. The levels of 10-dml and nbl produced by each combination of the PikAIII/PikAIV chimeras are shown as percents of the levels with wild-type (WT) PikAIII/PikAIV. The levels of starting material (thiophenol-pentaketide) consumed in each reaction were determined based on peak areas normalized to a control, which lacked enzyme. Domains are colored according to their source: PikAIII red, PikAIV magenta, CurG/CurH orange, CurK/CurL green. Catalytic and carrier domains are labeled circles, dimerization helices are boxes and ACPdd are rectangles, ddKS are crossed rectangles. ND, not detected. Data are presented as mean ± SD. See also Figure S4 and Table S1 and S2. Chemistry & Biology 2013 20, 1340-1351DOI: (10.1016/j.chembiol.2013.09.015) Copyright © 2013 Elsevier Ltd Terms and Conditions