Figure 2. Sfh5 crystal structure.

(A) Recombinant Sfh5 forms reddish brown rod-shaped crystals. Scale bar, 50 µm. (B) The asymmetric unit consists of three Sfh5 molecules and ribbon representations are colored by molecule. The bound heme is shown in ball and stick with carbon atoms colored in brown, oxygen – in red, and nitrogen in blue. (C) Ribbon diagram of the Sfh5 showing α-helices, loops and 3₁₀ turns in blue, and β-strands in red. A single non-covalently-bound heme b molecule is rendered in green with the brown sphere representing the heme iron. (D) The PtdIns binding substructure of Sec14-like PITPs is conserved in Sfh5. A PtdIns molecule was modeled into the closed Sfh5 structure by overlaying Sfh1::PtdIns crystal structure (PDB ID: 3B7N) onto the Sfh5 crystal structure. This binding model emphasizes the conserved interactions (dashed lines) between Sec14 family PtdIns-binding barcode residues of Sfh5 and elements of the PtdIns headgroup. The corresponding distances between PtdIns headgroup structural elements and side-chains of the barcode residues in this dock model are shown.

Figure 2—figure supplement 1. Structural features of Sfh5.

(A) Sfh5 crystal packing in the unit cell is shown. Each asymmetric unit within the unit cell is differentially colored for purpose of illustration. (B) Superposition of the Sfh5 (blue) and Sfh1 (silver) α-carbon backbones (rmsd 6.26 Å). Sfh1 represents the Sec14-like paralog most identical to Sec14 and it crystallizes in the closed conformation as does Sfh5. Bound PtdIns (Sfh1) and heme (Sfh5; pdb 3B7N) are omitted from the overlay. (C) Superposition of the Sfh5 (blue) and Sec14 (gold; pdb 1AUA) α-carbon backbones (rmsd 6.63 Å). Sec14 crystallizes as the open conformer. The displacement of the Sec14 gating helix relative to that of Sfh5 is highlighted and reflects the difference in atomic distance between reference residues K₁₉₂ and F₂₂₈ for Sfh5 and the Sec14 cognates R₁₉₅ and F₂₃₁. Bound β-octylglucoside (Sec14) and heme (Sfh5) are omitted from the overlay. (D) The Sfh5 G-module is a conformational switch element that consists of a series of short 3₁₀ helices organized into two distinct substructures, the β1-loop-β2 and the extended T5 loop linked to β5 (Ryan et al., 2007; Schaaf et al., 2008). Hydrogen bonds between these two elements contributes to opening and closing of the chamber gating helix. The close proximity of helices A7 (in blue) and A6 (in copper) indicate a ‘closed’ version of the protein. G₂₆₀, analogous to residue G₂₆₆ of the Sec14 G-module is shown as black sphere.