Hybrid Structure of a Dynamic Single-Chain Carboxylase from Deinococcus radiodurans  Anna Hagmann, Moritz Hunkeler, Edward Stuttfeld, Timm Maier  Structure  Volume 24, Issue 8, Pages 1227-1236 (August 2016) DOI: 10.1016/j.str.2016.06.001 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Dra YCC Domain Organization and Hexameric CT Domain Structure (A) Schematic domain organization of eukaryotic ACC, PCC, E. coli ACC, Map LCC, and Dra YCC. Domain boundaries of Dra YCC are indicated. BC is shown in blue, BCCP in yellow, CT in orange, and BT and CD domains in gray. (B) Top view of the hexameric CT domain organized as trimer of dimers. One dimer is depicted in orange and red, the remaining structure in gray; the D3 symmetry is indicated. N and C termini of the colored dimer are indicated with black spheres. (C) Side view of one CT dimer. The dimer exhibits an antiparallel arrangement with a central two-fold axis and active sites located distally at its interface. Disordered regions are indicated by dotted lines and the N and C lobes by brackets. (D) Dra YCC CT dimer with stabilizing C-terminal extensions highlighted as bold loops. Beginning of extensions and C terminus are indicated by spheres. View is the same as in (C), and only one dimer is shown for clarity. (E) Interactions of the conserved C-terminal DXW motif. View as indicated by the rectangle in (D); top protomer is clipped for clarity. Salt bridges are shown as black dashed lines. See also Figures S1 and S2. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Active Site of Dra YCC CT (A) The extended substrate binding cleft of the Dra YCC CT domain is lined with conserved residues. Color gradient from blue to red indicates increasing conservation; for visualization, a CoA moiety is modeled according to its positioning in the related MCC holoenzyme (PDB: 3U9R; Huang et al., 2010), and the carboxybiotin and palmitoyl are docked into active-site pockets. (B) Overlay of the CT active sites of M. tuberculosis (Mtb) ACC, Map LCC, and Dra YCC. Dra YCC CT is shown in surface representation (red and orange for two protomers); loops occluding the binding site tunnel of Mtb ACC and Map LCC are shown in yellow and blue, respectively (Reddy et al., 2014; Tran et al., 2015). See also Figure S3. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Dimerization and Active-Site Structure of the BC Domain (A) Cartoon representation of the dimeric BC domain structure. One protomer is shown in blue and the other in green; the two-fold symmetry dimer axis is indicated. The three subdomains are shown in shades of blue in one protomer. The lid in the B subdomain is in an open conformation and partially disordered. The active-site location is marked in one protomer. (B) The active-site structure of the Dra YCC BC domain superimposed onto E. coli ACC (PDB: 3G8C; Chou et al., 2008) in gray and Ruegeria pomeroyi PCC (PDB: 3N6R; Huang et al., 2010) in black. Catalytic residues are labeled; residue numbering is according to Dra YCC BC. Conservation of the BC active site indicates a conserved reaction mechanism of Dra YCC. See also Figure S4. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Negative-Stain EM Analysis of BC Domain Mobility (A) Cropped raw negative-stain EM micrograph. Three Dra YCC particles are indicated by white arrowheads. Scale bar, 500 Å. (B) Representative negative-stain EM 2D class averages of Dra YCC in top view (left), tilted view (middle), and side view (right). In the top view, the hexameric CT ring is clearly visible; the BC dimers are positioned laterally to the ring. (C) Projections of the Dra YCC CT crystal structure, filtered to 20-Å resolution, as well as images of the structure in gray, with one CT dimer shown in orange and red, corresponding to the class averages in (B). (D) Histogram of measured BC-CT distance in distance classes of 5-Å increments demonstrating the variable positioning of the BC domains relative to the CT domain hexamer. See also Figure S5. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 SAXS Analysis of Dra YCC (A) Comparison of experimental scattering curve (red), with calculated scattering curves for a symmetrical Dra YCC model based on Map LCC (green), the EOM-derived three-model Dra YCC ensemble (blue), and a single reconstituted asymmetric Dra YCC model (black, as shown in C). (B) Ensemble of Dra YCC models generated by EOM. (C) Reconstructed Dra YCC model featuring asymmetric placement of BC domains. The BCCP domains were modeled according to the crystal structure of the MCC BCCP (PDB: 3U9S; Huang et al., 2011). The BC dimer is colored blue and green, the CT monomers orange and red. The BCCP domains are colored yellow and the linkers gray. See also Table S1. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Dynamics and Flexibility of Carrier Protein-Dependent Multienzyme Families Rigid regions are shown in orange and red, while variable regions are shown in blue and green. Transparent domains show the approximate range of motion of the domains. Arrows represent example modes of domain motion. (A) Schematic representation of Dra YCC showing only a single instance of BCCP and BC dimer. (B) Schematic representation of E. coli. Only three E3 subunits are shown for clarity. Distances according to Murphy and Jensen (2005). (C) Schematic representation of the mammalian FAS with acyl carrier protein (ACP), thioesterase (TE) domain, and the linker connecting them to the enzyme (Maier et al., 2006). Only one ACP and TE are shown for clarity. Structure 2016 24, 1227-1236DOI: (10.1016/j.str.2016.06.001) Copyright © 2016 Elsevier Ltd Terms and Conditions