Open and closed conformations of two SpoIIAA-like proteins (YP_749275.1 and YP_001095227.1) provide insights into membrane association and ligand binding

Abstract

The crystal structures of the proteins encoded by the YP_749275.1 and YP_001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the `open' conformation (YP_001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the `closed' structure (YP_749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their `open' monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes.

Figure 1

Crystal structures of (a) YP_749275.1 (PDB code 2ook) and (b) YP_001095227.1 (PDB code 2q3l) shown as ribbon diagrams of protein monomers color-coded from the N-­terminus (blue) to the C-terminus (red). Helices α1–α4 and strands β1–β5 are indicated for YP_749275.1, while every tenth residue is numbered for YP_001095227.1. (c) Diagram showing the secondary-structure elements of YP_749275.1 and YP_001095227.1 superimposed on their primary sequences. Strands and helices are indicated by arrows and coils, respectively, and labeled sequentially as β1, β2 etc. and α1, α2 etc. Identical residues in these proteins are shown in white on a red background, while similar residues are shown as the reverse (red on a white background).

Figure 2

Structural comparison between YP_749275.1 and YP_001095227.1. (a) Superposition of the two structures underscores their overall similarity. The major differences arise from the different relative positions and orientations of helices α2 and α3. These two helices in YP_749275.1 (cyan) are close together, while they separate in YP_001095227.1 (green). (b) A surface representation of YP_001095227.1 in the ‘open’ state shows the presence of a wide cavity. The C atoms are colored grey, O atoms red and N atoms blue. The 2-methyl-2,4-pentanediol (MPD) molecules are shown as cyan and red sticks. The opening and closing of the cavity is regulated by the movement of helix α3 away from or towards α2, which involves an ∼5 Å translation and a 90° rotation of this helix. (c) YP_749275.1 reveals no cavity in the ‘closed’ state of the protein.

Figure 3

Dimerization modes. (a) Monomers in YP_749275.1 assemble with β-sheets lining up side by side to form a large, extended β-sheet. (b) YP_001095227.1 has a different dimerization mode with a more limited buried surface area. (c) Possible alternate mode of dimer formation in YP_001095227.1. Helices α2 and α3 are labeled.

Figure 4

Conserved residues in YP_749275.1 and YP_001095227.1. (a) A sequence alignment with other members of the PFAM PB000640 family (not shown) reveals several conserved residues (marked in grey boxes). Residues from the binding cavity are colored blue and residues that are predicted to penetrate to the lipid bilayer are colored red. These residues are indicated on the structure in (b) for YP_001095227.1 and (c) for YP_749275.1. The main cluster of conserved residues from strand β1 and helices α1 and α2 is shown in purple. The protein backbone is shown in a cartoon representation. The calculated membrane boundary is shown by grey dots. A few additional nonconserved residues involved in hydrophobic interactions (Leu8, Leu74 and Trp65) in YP_749275.1 are shown in orange.

Figure 5

Comparison of the putative membrane association of YP_001095227.1 and YP_749275.1 with that of the CRAL-TRIO domain of human α-tocopherol transfer protein (α-­TTP). (a) YP_001095227.1 in the ‘open’ conformation (PDB code 2q3l). (b) Dimeric form of YP_749275.1 in the ‘closed’ conformation (PDB code 2ook). The second molecule is colored blue and the lid helices enclosing the binding cavity are colored pink. Residues that move from the surface to the protein interior during the conformational switch are colored orange. (c) Human α-TTP in the ‘open’ conformation (PDB code 1oiz; Meier et al., 2003 ▶). The N-CRAL-TRIO domain is colored yellow and the lipid-binding CRAL-TRIO domain is colored green. Molecules of detergents or bound ligands are colored in dark green here and in (d). (d) α-TTP in the ‘closed’ conformation (PDB code 1r5l; Min et al., 2003 ▶). In all figures, residues that penetrate or are proposed to penetrate the lipid bilayer are colored purple. Calculated boundaries between lipid head groups and the acyl-chain region are shown by grey dots.