Crystal Structure of ARF1•Sec7 Complexed with Brefeldin A and Its Implications for the Guanine Nucleotide Exchange Mechanism  Elena Mossessova, Richard A Corpina, Jonathan Goldberg  Molecular Cell  Volume 12, Issue 6, Pages 1403-1411 (December 2003) DOI: 10.1016/S1097-2765(03)00475-1

Figure 1 Overall Structure of the ARF•GDP•Sec7•BFA Complex (A) Ribbon diagram with the Sec7 domain of Gea1 colored green and BFA colored blue with oxygen atoms red. Human ARF1 is orange, with its switch I and 2 regions in lighter hues. (B) “Open book” view of the interfacial surfaces, with space-filling models of the Sec7 domain and ARF1 rotated apart by 180°. Molecules are colored as in (A). Bound BFA is included on both proteins. Molecular Cell 2003 12, 1403-1411DOI: (10.1016/S1097-2765(03)00475-1)

Figure 2 Structural Basis for BFA Inhibition of GDP Release (A) The ARF1•GDP•Sec7•BFA complex is drawn and colored as in Figure 1A, but is rotated approximately 90° around a vertical axis; the view is along the superhelical axis of the Sec7 domain. BFA is colored blue. GDP is drawn as a ball-and-stick model, and Mg2+ is a gray sphere. (B) Structure of the nucleotide-free ARF1•Sec7 complex from a previous study (Goldberg, 1998). The Sec7 domain (of yeast Gea2) is oriented as in (A). The arrow indicates the direction of the conformational change in ARF1 that moves the nucleotide binding site toward residues of the Sec7 domain. (C) A close-up view of the ARF1•GDP•Sec7•BFA complex, oriented as in (A). Amino-acid residues of the Sec7 domain are labeled. 7.5 Å marks the closest contact distance between carboxylate oxygen atoms of the essential glutamate residue (E636) of the Sec7 domain and phosphate groups on GDP. (D) A close-up view of the nucleotide-free ARF1•Sec7 complex. Amino-acid residues of ARF1 are labeled. A model for GTP is included to indicate the position it occupies at the ARF1 active site. Note the overlap of the glutamate residue with the γ-phosphate group of GTP. Molecular Cell 2003 12, 1403-1411DOI: (10.1016/S1097-2765(03)00475-1)

Figure 3 Interactions between the Sec7 Domain and the Switch Regions of ARF1 Comparison of ARF1 within the ARF1•GDP•Sec7•BFA (colored blue) and nucleotide-free ARF1•Sec7 (orange) structures in the vicinity of the protein–protein interface. The two structures have been overlapped in a region encompassing the switch 1 and 2 elements (residues 45–84). The Sec7 domain is Gea1 from the ARF1•GDP•Sec7•BFA structure. (Note that Gea2 in the ARF1•Sec7 crystals [Goldberg, 1998] has a very similar structure). Amino-acid side chains are drawn for the key contact residues in switch 1 (labeled T48, I49, and F51) and switch 2 (I74, L77, H80, and Y81). Note the substantial positional change of the β2-β3 hairpin, but close overlap of the switch residues. Molecular Cell 2003 12, 1403-1411DOI: (10.1016/S1097-2765(03)00475-1)

Figure 4 Contact Residues at the BFA Binding Site (A) View in stereo toward the planar face of BFA. The carbon backbone of BFA is colored blue, with oxygen atoms red. Side chains of the Sec7 domain (green) and ARF1 (orange) residues that contact BFA are shown. Only residues of the Sec7 domain are labeled. Dotted red lines indicate hydrogen-bonding interactions. Water molecules are drawn as isolated red spheres. A difference electron density (SA omit [Brünger et al., 1998]) map at 2.4 Å resolution is drawn in yellow, contoured at 3 σ level. (B) View is rotated 90° from (A) around a horizontal axis. Only residues of ARF1 are labeled. The label 67(N) indicates the backbone amide nitrogen of ARF1 residue 67, which donates a hydrogen bond to a BFA hydroxyl group. Molecular Cell 2003 12, 1403-1411DOI: (10.1016/S1097-2765(03)00475-1)

Figure 5 A Possible Pathway for Conformational Rearrangement of ARF1 Catalyzed by the Sec7 Domain Space-filling models of four states of the ARF1 molecule, orientated with the Sec7 domain-interacting surface facing forward. Clockwise, (A) ARF1•GDP (Amor et al., 1994); (B) ARF1•GDP•Sec7•BFA from the present study, with the BFA molecule removed, as a putative mimic of an on-pathway intermediate (compare with Figure 1B); (C) nucleotide-free ARF1•Sec7 (Goldberg, 1998); (D) ARF1•GTP, from the structure of ARF1•GppNHp (Goldberg, 1998). In (B) and (C), the Sec7 domain is omitted. Majority of ARF1 is colored blue, switch 1 is yellow, and switch 2 is green. The key interfacial residues Phe51 (labeled F) and Tyr81 (Y) are indicated. Note that two tryptophan residues (W66 and W78) have rearranged in (C) to fill the cavity between Phe51 and Tyr81 present in (B). Molecular Cell 2003 12, 1403-1411DOI: (10.1016/S1097-2765(03)00475-1)