Structural Basis for Endosomal Targeting by the Bro1 Domain

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Structural Basis for Endosomal Targeting by the Bro1 Domain Jaewon Kim, Sujatha Sitaraman, Aitor Hierro, Bridgette M. Beach, Greg Odorizzi, James H. Hurley Developmental Cell Volume 8, Issue 6, Pages 937-947 (June 2005) DOI: 10.1016/j.devcel.2005.04.001 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Overall Structure of Bro1 (A) Electron density from MAD map contoured at 1.0 σ. (B and C) Two views of the overall structure. The N-terminal region is colored cyan; the N-terminal (non-TPR) portion of the helical solenoid is colored green; the β sheet is colored yellow; the TPR domain is colored magenta; and the C-terminal region is colored orange. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Homology of Bro1 Domains (A) Structural and sequence alignment of Bro1 domains. Key to symbols: red circle, TPR pocket residue; blue circle, electropositive region; magenta circles and squares, hydrophobic patch 1 and polar rim of hydrophobic patch 1, respectively; green circles and squares, hydrophobic patch 2 and polar rim of hydrophobic patch 2, respectively; black circle, buried polar cluster residues; black squares, buried polar residues stabilizing the N- or C-terminal regions, excluding TPR pocket residues. (B) Domain structures of selected Bro1 domain proteins. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 The TPR Domain Substructure (A and B) Structural overlay between Bro1 (magenta) and Hop1 (yellow), with the Hsp90 pentapeptide bound to Hop1 shown in green. (C) Details of interactions between the N terminus of Bro1 and the TPR-like pocket. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Functional Surfaces of the Bro1 Domain (A) Ribbon diagram of Bro1 domain shown in the same perspective as (B)–(D) for reference. (B) Molecular surface of the Bro1 domain colored according to residue property: green, hydrophobic; blue, basic; red, acidic; white, uncharged polar. (C) Bro1 domain surface colored according to residue conservation as in Figure 2A. (D) Bro1 domain surface colored according to electrostatic potential, with blue electropositive and red electronegative. (E) The same surface as in (D) is rotated by 180° about the x axis to show the opposite side of the boomerang. (F) Close-up of hydrophobic patch 1. (G) Close-up of hydrophobic patch 2. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The Bro1 Domain and Snf7 Form a Complex through Surface Patch 1 (A) His6-tagged Bro1 1–387 and untagged Snf7 were coexpressed in E. coli and copurify on Ni-NTA and gel filtration chromatography and visualized by SDS-PAGE and Coomassie staining. Bro1 1–387 mutants I144D and L336D in surface patch 1 express at normal levels, but coexpressed Snf7 does not copurify with them. (B) Ni-NTA agarose beads were used to isolate purified His6-tagged Snf7 or His6-tagged Vps20 that was mixed either with GST-tagged Bro1 1–387 or GST alone. (C) Glutathione-sepharose beads were used to isolate either GST-tagged Bro1 1–387 or GST alone that had been mixed with a total yeast extract. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The Bro1 Domain Mediates Bro1 Localization The indicated strains were stained with FM 4-64 and examined by fluorescence and DIC microscopy. Arrowheads indicate class E compartments. Scale bars equal 2 μm. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 The Bro1 Domain and Surface Patch 1 Are Required for Cargo Sorting The indicated strains expressing GFP-CPS were examined by fluorescence and DIC microscopy. Scale bar equals 2 μm. Developmental Cell 2005 8, 937-947DOI: (10.1016/j.devcel.2005.04.001) Copyright © 2005 Elsevier Inc. Terms and Conditions