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Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate

View Article: PubMed Central - PubMed

ABSTRACT

Energy coupling factor (ECF) transporters are responsible for the uptake of essential scarce nutrients in prokaryotes. This ATP-binding cassette transporter family comprises two subgroups that share a common architecture forming a tripartite membrane protein complex consisting of a translocation component and ATP hydrolyzing module and a substrate-capture (S) component. Here, we present the crystal structure of YkoE from Bacillus subtilis, the S component of the previously uncharacterized group I ECF transporter YkoEDC. Structural and biochemical analyses revealed the constituent residues of the thiamine-binding pocket as well as an unexpected mode of vitamin recognition. In addition, our experimental and bioinformatics data demonstrate major differences between YkoE and group II ECF transporters and indicate how group I vitamin transporter S components have diverged from other group I and group II ECF transporters.

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Structure Superposition of S Components(A) Superposition of the structure of YkoE (dark blue) and the structures of group II S components colored as follows: FolT with bound folate (PDB: 4Z7F) in pink, RibU (PDB: 3P5N) in light green, HMP (PDB: 4HZU) in magenta, ThiT (PDB: 3RLB) in yellow, apoFolT (PDB: 4HUQ) in gray, BioY (PDB: 4DVE) in green, PanT (PDB: 4RFS) in orange.(B) Superposition of YkoE (dark blue) and NikM (light pink).(C) Structure-based multiple sequence alignment of group I and II S components. The secondary structural elements of YkoE are shown on the top. Structurally equivalent residues are shown in uppercase. The residues contributing to the conserved ΦXXXA motif are indicated with red dots at the bottom of the alignment, the YkoE residues that interact with the thiamine are indicated with yellow dots, and the Phe residues that obscure the groove with blue dots.
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fig2: Structure Superposition of S Components(A) Superposition of the structure of YkoE (dark blue) and the structures of group II S components colored as follows: FolT with bound folate (PDB: 4Z7F) in pink, RibU (PDB: 3P5N) in light green, HMP (PDB: 4HZU) in magenta, ThiT (PDB: 3RLB) in yellow, apoFolT (PDB: 4HUQ) in gray, BioY (PDB: 4DVE) in green, PanT (PDB: 4RFS) in orange.(B) Superposition of YkoE (dark blue) and NikM (light pink).(C) Structure-based multiple sequence alignment of group I and II S components. The secondary structural elements of YkoE are shown on the top. Structurally equivalent residues are shown in uppercase. The residues contributing to the conserved ΦXXXA motif are indicated with red dots at the bottom of the alignment, the YkoE residues that interact with the thiamine are indicated with yellow dots, and the Phe residues that obscure the groove with blue dots.

Mentions: To gain insights into the function of the YkoEDC ECF transporter, we solved the crystal structure of its S component YkoE. The gene was cloned from several bacterial species, and the protein was expressed and purified to homogeneity. YkoE failed to crystallize using the traditional vapor-diffusion methods after screening several different homologs. However, YkoE from Bacillus subtilis could be readily crystallized using the lipidic cubic phase (LCP) method. The structure was solved using single-wavelength anomalous dispersion (SAD) with selenomethionine-labeled YkoE to 1.95 Å resolution. The electron density from native crystals was of sufficient quality to build the entire molecule of YkoE with the exception of the four N-terminal amino acids (Figure S1A). The structure of YkoE revealed a six helical transmembrane domain with the overall fold reminiscent of S components from group II ECF transporters (root-mean-square deviation between YkoE and other S components ranges between 2.6 and 3.6 Å) (Figures 1C and 2A). YkoE possesses an additional C-terminal helix that presumably protrudes toward the cytosol and lies perpendicular to the lipid bilayer (Figure 1D). The present orientation of the helix is likely stabilized by the crystallographic contacts between neighboring molecules (Figure S1B). The six hydrophobic helices form a tight fold with an open cavity with a volume of 545 Å3 facing the extracellular part of the membrane. The most conserved amino acid residues in YkoE map to the interior of the cavity as well as residues involved in the interhelical packing within the molecule (Figure 3A). In YkoE, helix H1 is highly extended with a bend in the middle, leading into a sharp turn joining to helix H2 (Figure 3B). Helix H2 possesses a conserved Pro44 that breaks up the α-helical backbone, giving rise to a kink in the helix that leads into a 310 helical conformation, returning to a regular α-helical backbone after a short amino acid stretch (Figures 1D and 3B). Such a structural feature is reminiscent of helix H4 in ThiT where the π bulge dictates the conformation of the residues forming the thiamine-binding site (Erkens et al., 2011). In YkoE, helix H2 packs very tightly against helix H6, which bears a highly conserved π bulge that introduces an additional kink at the nearly invariant Gly47 residue in helix H2, and thus reversing the 310 helical stretch to an α-helical one (Figure 3B). This packing arrangement, together with the surrounding helices H3, H4, and H5, creates a funnel-like substrate-binding cavity.


Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate
Structure Superposition of S Components(A) Superposition of the structure of YkoE (dark blue) and the structures of group II S components colored as follows: FolT with bound folate (PDB: 4Z7F) in pink, RibU (PDB: 3P5N) in light green, HMP (PDB: 4HZU) in magenta, ThiT (PDB: 3RLB) in yellow, apoFolT (PDB: 4HUQ) in gray, BioY (PDB: 4DVE) in green, PanT (PDB: 4RFS) in orange.(B) Superposition of YkoE (dark blue) and NikM (light pink).(C) Structure-based multiple sequence alignment of group I and II S components. The secondary structural elements of YkoE are shown on the top. Structurally equivalent residues are shown in uppercase. The residues contributing to the conserved ΦXXXA motif are indicated with red dots at the bottom of the alignment, the YkoE residues that interact with the thiamine are indicated with yellow dots, and the Phe residues that obscure the groove with blue dots.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037267&req=5

fig2: Structure Superposition of S Components(A) Superposition of the structure of YkoE (dark blue) and the structures of group II S components colored as follows: FolT with bound folate (PDB: 4Z7F) in pink, RibU (PDB: 3P5N) in light green, HMP (PDB: 4HZU) in magenta, ThiT (PDB: 3RLB) in yellow, apoFolT (PDB: 4HUQ) in gray, BioY (PDB: 4DVE) in green, PanT (PDB: 4RFS) in orange.(B) Superposition of YkoE (dark blue) and NikM (light pink).(C) Structure-based multiple sequence alignment of group I and II S components. The secondary structural elements of YkoE are shown on the top. Structurally equivalent residues are shown in uppercase. The residues contributing to the conserved ΦXXXA motif are indicated with red dots at the bottom of the alignment, the YkoE residues that interact with the thiamine are indicated with yellow dots, and the Phe residues that obscure the groove with blue dots.
Mentions: To gain insights into the function of the YkoEDC ECF transporter, we solved the crystal structure of its S component YkoE. The gene was cloned from several bacterial species, and the protein was expressed and purified to homogeneity. YkoE failed to crystallize using the traditional vapor-diffusion methods after screening several different homologs. However, YkoE from Bacillus subtilis could be readily crystallized using the lipidic cubic phase (LCP) method. The structure was solved using single-wavelength anomalous dispersion (SAD) with selenomethionine-labeled YkoE to 1.95 Å resolution. The electron density from native crystals was of sufficient quality to build the entire molecule of YkoE with the exception of the four N-terminal amino acids (Figure S1A). The structure of YkoE revealed a six helical transmembrane domain with the overall fold reminiscent of S components from group II ECF transporters (root-mean-square deviation between YkoE and other S components ranges between 2.6 and 3.6 Å) (Figures 1C and 2A). YkoE possesses an additional C-terminal helix that presumably protrudes toward the cytosol and lies perpendicular to the lipid bilayer (Figure 1D). The present orientation of the helix is likely stabilized by the crystallographic contacts between neighboring molecules (Figure S1B). The six hydrophobic helices form a tight fold with an open cavity with a volume of 545 Å3 facing the extracellular part of the membrane. The most conserved amino acid residues in YkoE map to the interior of the cavity as well as residues involved in the interhelical packing within the molecule (Figure 3A). In YkoE, helix H1 is highly extended with a bend in the middle, leading into a sharp turn joining to helix H2 (Figure 3B). Helix H2 possesses a conserved Pro44 that breaks up the α-helical backbone, giving rise to a kink in the helix that leads into a 310 helical conformation, returning to a regular α-helical backbone after a short amino acid stretch (Figures 1D and 3B). Such a structural feature is reminiscent of helix H4 in ThiT where the π bulge dictates the conformation of the residues forming the thiamine-binding site (Erkens et al., 2011). In YkoE, helix H2 packs very tightly against helix H6, which bears a highly conserved π bulge that introduces an additional kink at the nearly invariant Gly47 residue in helix H2, and thus reversing the 310 helical stretch to an α-helical one (Figure 3B). This packing arrangement, together with the surrounding helices H3, H4, and H5, creates a funnel-like substrate-binding cavity.

View Article: PubMed Central - PubMed

ABSTRACT

Energy coupling factor (ECF) transporters are responsible for the uptake of essential scarce nutrients in prokaryotes. This ATP-binding cassette transporter family comprises two subgroups that share a common architecture forming a tripartite membrane protein complex consisting of a translocation component and ATP hydrolyzing module and a substrate-capture (S) component. Here, we present the crystal structure of YkoE from Bacillus subtilis, the S component of the previously uncharacterized group I ECF transporter YkoEDC. Structural and biochemical analyses revealed the constituent residues of the thiamine-binding pocket as well as an unexpected mode of vitamin recognition. In addition, our experimental and bioinformatics data demonstrate major differences between YkoE and group II ECF transporters and indicate how group I vitamin transporter S components have diverged from other group I and group II ECF transporters.

No MeSH data available.


Related in: MedlinePlus