Chao WC , Wade BO , Bouchoux C , Jones AW , Purkiss AG , Federico S , O'Reilly N , Snijders AP , Uhlmann F , Singleton MR .

FC001155 Cancer Research UK, FC001198 Cancer Research UK, Medical Research Council , Wellcome Trust , 19343 Cancer Research UK

	Figure 2. Structures of the xEco2-K106-CoA and xEco2-K105-CoA complexes.(A) Schematic of xEco2 indicating the zinc-finger (ZnF) domain and the construct boundary of the acetyltransferase (ACT) domain. PIP box – PCNA interaction peptide, PBM-A/B as defined in Higashi et al.27. (B) Design of the Eco1 substrate with a 13-residue Smc3 peptide conjugated with CoA at K105 (K105-CoA). (C) Design of the Eco1 substrate with a 13-residue Smc3 peptide conjugated with CoA at K106 (K106-CoA). (D) Structure of the xEco2 ACT. The xEco2 ACT consists of an N-terminal domain (cyan) with a central β hairpin (orange) and a C-terminal domain (wheat) with a conserved C extension (red). (E) Structure of the xEco2 ACT bound to K105-CoA (yellow). The K105-CoA peptide adopts a β turn conformation and is coordinated by the C extension as a contiguous β sheet. The xEco2 ACT has the same colouring scheme as in (D). (F) Structure of the xEco2 ACT bound to K106-CoA (yellow). The K106-CoA peptide is coordinated in a β strand conformation by the β hairpin. The xEco2 ACT has the same colouring scheme as in (D) with the C extension (red) positioning upwards to allow the full accommodation of the K106-CoA peptide. (G) Yeast survival assays comparing wild-type and mutant Eco1 strains. ECO1 mutants were tested for the ability to restore viability to a strain carrying an ECO1 degron allele. Cells of each strain were serially diluted and spotted on synthetic minimal medium without methionine (CSM – Met) plates or on plates containing indoleacetic acid (IAA). (H) In-vivo acetylation assays using anti-acetyl Smc3 antibody. Expression levels of Eco1 mutants were monitored by anti-HA antibody. Western blots have been cropped for presentation.
	Figure 3. Substrate binding and conformational changes of the central β hairpin and C extension.(A) Details of xEco2-K105-CoA interaction showing substrate hairpin (yellow) coordinated by the C extension (red) in form of a β sheet. (B) Conserved surface rendition of K105-CoA bound to xEco2. (C) Details of xEco2-K106-CoA interaction showing substrate peptide (yellow) coordinated by the central β hairpin (orange) in form of a β sheet. (D) Conserved surface rendition of K106-CoA bound to xEco2. (E) Superimposition of peptide-free (red), K105-CoA (green), and K106-CoA (salmon) bound xEco2 structures showing occupation of and displacement of W623 from the central hydrophobic pocket. (F) Superimposition of the C extension in peptide-free (red), K105-CoA (green), and K106-CoA (salmon) bound conformations. The C extension shows a 180° flip from coordinating W623 at the hydrophobic pocket to an open conformation upon K106-CoA binding.
	Figure 4. Mechanism of Eco1-mediated Smc3 acetylation.(A) Speculative model for Eco1-Smc3 interaction created by docking of xEco2-K105-CoA structure (salmon and blue) onto the S. cerevisiae Smc3-Scc1 (Smc3 is green; Scc1 is cyan) structure with reference to the relative positions of the tandem lysines (S. cerevisiae K112 and K113). (B) Speculative model for Eco1-Smc3 interaction created by docking of xEco2-K106-CoA structure (salmon and blue) onto the S. cerevisiae Smc3-Scc1 (Smc3 is green; Scc1 is cyan) structure with reference to the relative positions of the tandem lysines (S. cerevisiae K112 and K113). xEco2 rotates by 180° along the plane of the β sheet of Smc3 ATPase domain. (C) Schematics of Eco1 substrate specificity conferred by the concerted conformational changes of the central β hairpin (orange) and the C extension (red). The conserved hydrophobic pocket is occupied by W623 of xEco2 in the peptide-free and K105 targeting configurations, and by Y109 from Smc3 during K106 targeting respectively.

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