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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.

No MeSH data available.


Structural Overview of the Conserved Motifs in the Group I S Component YkoE(A) Substrate cavity and interhelical contacts are the most conserved regions of YkoE. Conservation of amino acid residues was analyzed using ConSurf (Ashkenazy et al., 2010). 980 non-redundant sequences of YkoE homologs were used in the alignment to emphasize the most conserved regions of the structure. Highly conserved residues are depicted as burgundy patches; moderately conserved side chains are shown in light pink. Weakly conserved residues are colored in cyan. Residues that exhibit some degree of conservation among the 980 homologs of YkoE are in white.(B) Ribbon representation of the packing conformations of helix H2 and helix H6. Helix H2 is comprised of α-310-α helical elements that allow it to pack tightly against helix H6, thereby closing the cavity from the cytoplasmic side. The highly conserved π bulge is in the middle of helix H6.
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fig3: Structural Overview of the Conserved Motifs in the Group I S Component YkoE(A) Substrate cavity and interhelical contacts are the most conserved regions of YkoE. Conservation of amino acid residues was analyzed using ConSurf (Ashkenazy et al., 2010). 980 non-redundant sequences of YkoE homologs were used in the alignment to emphasize the most conserved regions of the structure. Highly conserved residues are depicted as burgundy patches; moderately conserved side chains are shown in light pink. Weakly conserved residues are colored in cyan. Residues that exhibit some degree of conservation among the 980 homologs of YkoE are in white.(B) Ribbon representation of the packing conformations of helix H2 and helix H6. Helix H2 is comprised of α-310-α helical elements that allow it to pack tightly against helix H6, thereby closing the cavity from the cytoplasmic side. The highly conserved π bulge is in the middle of helix H6.

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
Structural Overview of the Conserved Motifs in the Group I S Component YkoE(A) Substrate cavity and interhelical contacts are the most conserved regions of YkoE. Conservation of amino acid residues was analyzed using ConSurf (Ashkenazy et al., 2010). 980 non-redundant sequences of YkoE homologs were used in the alignment to emphasize the most conserved regions of the structure. Highly conserved residues are depicted as burgundy patches; moderately conserved side chains are shown in light pink. Weakly conserved residues are colored in cyan. Residues that exhibit some degree of conservation among the 980 homologs of YkoE are in white.(B) Ribbon representation of the packing conformations of helix H2 and helix H6. Helix H2 is comprised of α-310-α helical elements that allow it to pack tightly against helix H6, thereby closing the cavity from the cytoplasmic side. The highly conserved π bulge is in the middle of helix H6.
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Related In: Results  -  Collection

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fig3: Structural Overview of the Conserved Motifs in the Group I S Component YkoE(A) Substrate cavity and interhelical contacts are the most conserved regions of YkoE. Conservation of amino acid residues was analyzed using ConSurf (Ashkenazy et al., 2010). 980 non-redundant sequences of YkoE homologs were used in the alignment to emphasize the most conserved regions of the structure. Highly conserved residues are depicted as burgundy patches; moderately conserved side chains are shown in light pink. Weakly conserved residues are colored in cyan. Residues that exhibit some degree of conservation among the 980 homologs of YkoE are in white.(B) Ribbon representation of the packing conformations of helix H2 and helix H6. Helix H2 is comprised of α-310-α helical elements that allow it to pack tightly against helix H6, thereby closing the cavity from the cytoplasmic side. The highly conserved π bulge is in the middle of helix H6.
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.