Structure of the ACF7 EF-Hand-GAR Module and Delineation of Microtubule Binding Determinants  Thomas R. Lane, Elaine Fuchs, Kevin C. Slep  Structure  Volume 25, Issue 7, Pages 1130-1138.e6 (July 2017) DOI: 10.1016/j.str.2017.05.006 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1130-1138.e6DOI: (10.1016/j.str.2017.05.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Architecture of the ACF7 EF1-EF2-GAR Module (A and B) hACF7 domain architecture (A) and zoom view (B) of the MT-binding region. (C) Sequence alignment of the EF1-EF2-GAR module from human, Danio rerio, Drosophila melanogaster, and C. elegans spectraplakins, and human Gas2. hACF7 EF1-EF2-GAR 2° structure and residue number are depicted above, residues mutated are indicated below. (D) SEC-MALS analysis of the EF1-EF2-GAR module. The molecular weight of a monomer is indicated by the dashed line. The peak measured accounts for 96.4% of the total mass eluted. (E) Model of the EF1-EF2-GAR modules observed in the ASU. See also Figure S1. Structure 2017 25, 1130-1138.e6DOI: (10.1016/j.str.2017.05.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 EF1-EF2 Domain Structure (A–C) Structure of the EF1-EF2 domain in different orientations. Following the N-terminal α-helix (cyan) are the EF1 (yellow) and EF2 (orange) helix-turn-helix motifs. Calcium atoms are shown in green. (A) View down the 2-fold pseudo symmetry axis. (B) Side view of EF-1. (C) EF-1 and EF-2 straddle the N-terminal α1 helix. (D) Secondary structure topology of the EF1-EF2 domain showing the location of residues that coordinate calcium (green asterisks). (E and F) The EF1-EF2 domain illustrating cross-species conservation as delineated in Figure 1C (above, spherical representation) and displaying electrostatic surface potential (below, surface representation). Domain orientation in (E) and (F) corresponds to the orientation in (B) and (C), respectively. (G and H) Zoom views of the EF1 and EF2 calcium-binding sites. Canonical EF-Hand residues are indicated. See also Figure S2. Structure 2017 25, 1130-1138.e6DOI: (10.1016/j.str.2017.05.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The GAR Domain Is a Novel α/β Sandwich that Coordinates Zinc (A) Secondary structure topology of the GAR domain α/β sandwich showing the location of the residues that coordinate zinc. (B) Ribbon diagram of the GAR domain as colored in (A). (C) The GAR domain as shown in (B), rotated 180° about the y axis. (D) Zoom view of the zinc-binding site boxed in (B), showing the Cys2-Asp-Cys residues that coordinate zinc. (E and F) The GAR domain illustrating cross-species conservation as delineated in Figure 1C (above, spherical representation) and displaying electrostatic surface potential (below, surface representation). Domain orientation in (E) and (F) corresponds to the orientation in (B) and (C) respectively. (G and H) Zoom view of two positively charged regions on the GAR domain, corresponding to the boxed regions in (B) and (C), respectively. See also Figure S2. Structure 2017 25, 1130-1138.e6DOI: (10.1016/j.str.2017.05.006) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 The GAR β3-β5 Basic Region Mediates MT Binding (A) MT co-sedimentation assays of WT and mutant EF1-EF2-GAR constructs. (B–H) Live-cell analysis of GFP-EF1-EF2-GAR WT and mutant constructs in HEK293 cells. (B′–H′) Analysis of GFP-EF1-EF2-GAR WT and mutant constructs for MT co-localization in fixed HEK293 cells. All scale bars, 20 μm. See also Figures S3 and S4. Structure 2017 25, 1130-1138.e6DOI: (10.1016/j.str.2017.05.006) Copyright © 2017 Elsevier Ltd Terms and Conditions