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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.


Side by Side Comparison of YkoE and ThiTBoth proteins are colored by conservation.(A) YkoE (left) and ThiT (right) with bound thiamine.(B) The constituent residues of the YkoE (left) and ThiT (right) thiamine binding pocket that interact with thiamine.
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fig5: Side by Side Comparison of YkoE and ThiTBoth proteins are colored by conservation.(A) YkoE (left) and ThiT (right) with bound thiamine.(B) The constituent residues of the YkoE (left) and ThiT (right) thiamine binding pocket that interact with thiamine.

Mentions: During the initial stages of refinement, the density for thiamine became apparent and allowed the modeling of the full molecule unambiguously (Figure 4A). The thiamine molecule is present at the base of the cavity found in the extracellular part of the membrane (Figures 4B and 4C). The pyrimidine group forms π-stacking interactions with a highly conserved Trp49 located at the kink of helix H2. In addition, the pyrimidine group is coordinated by H bonds by highly conserved Glu77 and Gln95 residues located on helix H3 and H4, respectively. The thiazole ring of thiamine forms H-bonding interactions with Asp131 and Tyr46 (Figure 4D). The residues coordinating the pyrimidine moiety of thiamine are more conserved than those coordinating the thiazole moiety (Figure S2A). The orientation of the thiamine in the YkoE binding pocket differs significantly from that of the thiamine bound to ThiT, a group II ECF S component (Erkens et al., 2011). The thiazole moiety of thiamine in ThiT points to the bottom of the binding pocket and the pyrimidine moiety faces the extracellular side (Figures 5A and S3). In contrast, the thiamine bound to YkoE is in a reverse orientation and located much deeper in the binding pocket. There are also differences between the key interactions for thiamine binding in the YkoE and ThiT binding pockets. In ThiT, the thiazole ring is sandwiched between the conserved Trp34 and His125 located on loop L1 and helix H5, respectively. In addition, the Glu84 residue in helix H4 forms a hydrogen bond with the pyrimidine moiety and Trp133 located at the cap of helix H5 makes a stacking interaction (Figure 5B). The latter is reminiscent of the interaction between the conserved Trp49 and the pyrimidine ring of the thiamine in the YkoE structure. The conformation of the thiamine molecule in the binding sites of YkoE and ThiT is almost identical, both molecules having the low-energy F conformation as defined by the dihedral angles ϕT (C5′-C3,5′-N3-C2) and ϕP (N3-C3,5′-C5′-C4′) (Pletcher et al., 1977). The thiamine-binding crevice in YkoE is open and not protected by lid closure mediated by loop L1 as observed for several group II ECF S components (Figures 4B and S3) (Zhang et al., 2010, Erkens et al., 2011, Zhao et al., 2015).


Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate
Side by Side Comparison of YkoE and ThiTBoth proteins are colored by conservation.(A) YkoE (left) and ThiT (right) with bound thiamine.(B) The constituent residues of the YkoE (left) and ThiT (right) thiamine binding pocket that interact with thiamine.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig5: Side by Side Comparison of YkoE and ThiTBoth proteins are colored by conservation.(A) YkoE (left) and ThiT (right) with bound thiamine.(B) The constituent residues of the YkoE (left) and ThiT (right) thiamine binding pocket that interact with thiamine.
Mentions: During the initial stages of refinement, the density for thiamine became apparent and allowed the modeling of the full molecule unambiguously (Figure 4A). The thiamine molecule is present at the base of the cavity found in the extracellular part of the membrane (Figures 4B and 4C). The pyrimidine group forms π-stacking interactions with a highly conserved Trp49 located at the kink of helix H2. In addition, the pyrimidine group is coordinated by H bonds by highly conserved Glu77 and Gln95 residues located on helix H3 and H4, respectively. The thiazole ring of thiamine forms H-bonding interactions with Asp131 and Tyr46 (Figure 4D). The residues coordinating the pyrimidine moiety of thiamine are more conserved than those coordinating the thiazole moiety (Figure S2A). The orientation of the thiamine in the YkoE binding pocket differs significantly from that of the thiamine bound to ThiT, a group II ECF S component (Erkens et al., 2011). The thiazole moiety of thiamine in ThiT points to the bottom of the binding pocket and the pyrimidine moiety faces the extracellular side (Figures 5A and S3). In contrast, the thiamine bound to YkoE is in a reverse orientation and located much deeper in the binding pocket. There are also differences between the key interactions for thiamine binding in the YkoE and ThiT binding pockets. In ThiT, the thiazole ring is sandwiched between the conserved Trp34 and His125 located on loop L1 and helix H5, respectively. In addition, the Glu84 residue in helix H4 forms a hydrogen bond with the pyrimidine moiety and Trp133 located at the cap of helix H5 makes a stacking interaction (Figure 5B). The latter is reminiscent of the interaction between the conserved Trp49 and the pyrimidine ring of the thiamine in the YkoE structure. The conformation of the thiamine molecule in the binding sites of YkoE and ThiT is almost identical, both molecules having the low-energy F conformation as defined by the dihedral angles ϕT (C5′-C3,5′-N3-C2) and ϕP (N3-C3,5′-C5′-C4′) (Pletcher et al., 1977). The thiamine-binding crevice in YkoE is open and not protected by lid closure mediated by loop L1 as observed for several group II ECF S components (Figures 4B and S3) (Zhang et al., 2010, Erkens et al., 2011, Zhao et al., 2015).

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.