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Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters.

Zhao Q, Wang C, Wang C, Guo H, Bao Z, Zhang M, Zhang P - Nat Commun (2015)

Bottom Line: Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling.Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity.Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

No MeSH data available.


Overall structure of EfFolT.(a) Overall structure of EfFolT in ribbon cartoon. The six transmembrane helices 1–6 (SM1-6) are coloured from blue to red. The bound substrate folate is shown with a stick model coloured in magenta. (b) A cross-section drawing of EfFolT shows the folate-binding pocket (blue and red colours represent positive and negative charges, respectively). (c) Mass spectrometry results of the substrate bound with EfFolT. Lower panel shows the folate standard; upper panel shows the substrate bound with EfFolT.
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f1: Overall structure of EfFolT.(a) Overall structure of EfFolT in ribbon cartoon. The six transmembrane helices 1–6 (SM1-6) are coloured from blue to red. The bound substrate folate is shown with a stick model coloured in magenta. (b) A cross-section drawing of EfFolT shows the folate-binding pocket (blue and red colours represent positive and negative charges, respectively). (c) Mass spectrometry results of the substrate bound with EfFolT. Lower panel shows the folate standard; upper panel shows the substrate bound with EfFolT.

Mentions: There are small conformational differences among the six molecules (root mean square deviations/RMSD of 0.7–1.2 Å). The overall structure of EfFolT is similar to the reported structures of EcfS proteins (RMSD of 1.5–3.3 Å) which consist of six transmembrane helices (SM1-6, transmembrane helices 1–6 of EcfS) that form a helix bundle. The N-terminus and C-terminus are both at the cytoplasmic side. Three loops L1, L3, L5 connect SM1-SM2, SM3-SM4 and SM5-SM6, respectively, from the periplasmic side (Fig. 1a). A pocket with the depth of 24 Å and volume of 1,230 Å3 is formed by the six transmembrane helices near the periplasmic side and is covered by the L1 loop from top (Fig. 1b). The substrate bound in the pocket is verified by mass spectrometry to be folate (Fig. 1c) and further confirmed by the electron density map in the pocket (Fig. 2a).


Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters.

Zhao Q, Wang C, Wang C, Guo H, Bao Z, Zhang M, Zhang P - Nat Commun (2015)

Overall structure of EfFolT.(a) Overall structure of EfFolT in ribbon cartoon. The six transmembrane helices 1–6 (SM1-6) are coloured from blue to red. The bound substrate folate is shown with a stick model coloured in magenta. (b) A cross-section drawing of EfFolT shows the folate-binding pocket (blue and red colours represent positive and negative charges, respectively). (c) Mass spectrometry results of the substrate bound with EfFolT. Lower panel shows the folate standard; upper panel shows the substrate bound with EfFolT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525288&req=5

f1: Overall structure of EfFolT.(a) Overall structure of EfFolT in ribbon cartoon. The six transmembrane helices 1–6 (SM1-6) are coloured from blue to red. The bound substrate folate is shown with a stick model coloured in magenta. (b) A cross-section drawing of EfFolT shows the folate-binding pocket (blue and red colours represent positive and negative charges, respectively). (c) Mass spectrometry results of the substrate bound with EfFolT. Lower panel shows the folate standard; upper panel shows the substrate bound with EfFolT.
Mentions: There are small conformational differences among the six molecules (root mean square deviations/RMSD of 0.7–1.2 Å). The overall structure of EfFolT is similar to the reported structures of EcfS proteins (RMSD of 1.5–3.3 Å) which consist of six transmembrane helices (SM1-6, transmembrane helices 1–6 of EcfS) that form a helix bundle. The N-terminus and C-terminus are both at the cytoplasmic side. Three loops L1, L3, L5 connect SM1-SM2, SM3-SM4 and SM5-SM6, respectively, from the periplasmic side (Fig. 1a). A pocket with the depth of 24 Å and volume of 1,230 Å3 is formed by the six transmembrane helices near the periplasmic side and is covered by the L1 loop from top (Fig. 1b). The substrate bound in the pocket is verified by mass spectrometry to be folate (Fig. 1c) and further confirmed by the electron density map in the pocket (Fig. 2a).

Bottom Line: Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling.Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity.Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

View Article: PubMed Central - PubMed

Affiliation: National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

No MeSH data available.