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Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter.

Lee Y, Nishizawa T, Yamashita K, Ishitani R, Nureki O - Nat Commun (2015)

Bottom Line: A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer.The conserved PQ-loop motif serves as a molecular hinge that enables the 'binder clip-like' motion of SemiSWEET.The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
SWEET family proteins mediate sugar transport across biological membranes and play crucial roles in plants and animals. The SWEETs and their bacterial homologues, the SemiSWEETs, are related to the PQ-loop family, which is characterized by highly conserved proline and glutamine residues (PQ-loop motif). Although the structures of the bacterial SemiSWEETs were recently reported, the conformational transition and the significance of the conserved motif in the transport cycle have remained elusive. Here we report crystal structures of SemiSWEET from Escherichia coli, in the both inward-open and outward-open states. A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer. The conserved PQ-loop motif serves as a molecular hinge that enables the 'binder clip-like' motion of SemiSWEET. The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

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Structure and function of SemiSWEET.(a) Time course of [14C]-sucrose uptake by proteoliposomes containing EcSemiSWEET (solid black squares) or empty control liposomes (open black squares) (mean± s.e.m., n=6). (b) Plots of the sucrose uptake rate versus the extra-liposomal sucrose concentration (mean±s.e.m., n=3). (c) Overall structure of the inward-open SemiSWEET dimer, viewed parallel to the membrane (upper) or from the intracellular side (lower). In the ribbon representations (left), TM1, TM2, and TM3 of one protomer are coloured blue, green and red, respectively, and the other protomer is coloured grey. In the surface representations (right), the dimer is coloured according to the surface electrostatic potential. The cut-away surface is shown in the parallel view. The twofold axis is indicated by dashed lines and an almond-shaped symbol. (d) Overall structure of the outward-open SemiSWEET dimer, viewed parallel to the membrane (upper) and from the extracellular side (lower), coloured as in c.
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f1: Structure and function of SemiSWEET.(a) Time course of [14C]-sucrose uptake by proteoliposomes containing EcSemiSWEET (solid black squares) or empty control liposomes (open black squares) (mean± s.e.m., n=6). (b) Plots of the sucrose uptake rate versus the extra-liposomal sucrose concentration (mean±s.e.m., n=3). (c) Overall structure of the inward-open SemiSWEET dimer, viewed parallel to the membrane (upper) or from the intracellular side (lower). In the ribbon representations (left), TM1, TM2, and TM3 of one protomer are coloured blue, green and red, respectively, and the other protomer is coloured grey. In the surface representations (right), the dimer is coloured according to the surface electrostatic potential. The cut-away surface is shown in the parallel view. The twofold axis is indicated by dashed lines and an almond-shaped symbol. (d) Overall structure of the outward-open SemiSWEET dimer, viewed parallel to the membrane (upper) and from the extracellular side (lower), coloured as in c.

Mentions: We screened the bacterial SemiSWEETs and identified E. coli SemiSWEET (EcSemiSWEET) as a suitable candidate for structural studies. EcSemiSWEET possesses the conserved Pro-Gln motif and shares 36% sequence identity and 57% similarity with Bradyrhizobium japonicum SemiSWEET, which was previously characterized as a sucrose uniporter (Supplementary Fig. 1)24. To test the sucrose transport activity of EcSemiSWEET, we reconstituted the purified protein into liposomes and measured [14C]-sucrose uptake into the proteoliposomes. The proteoliposomes containing EcSemiSWEET showed slow but significant [14C]-sucrose uptake, as compared with the control empty liposomes (Fig. 1a). The unusually slow uptake of EcSemiSWEET compared with those of eukaryotic SWEET transporters suggests that sucrose might not be the physiological substrate. Nonetheless, our transport assay confirmed the sucrose transport activity of EcSemiSWEET. The rate of [14C]-sucrose uptake increased linearly according to the extraliposomal sucrose concentration and was not saturated even up to 300 mM, indicating the low-affinity binding of EcSemiSWEET to sucrose (Fig. 1b). Previous studies also reported the low affinities of the plant SWEETs for sugars1314, suggesting a common transport mechanism.


Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter.

Lee Y, Nishizawa T, Yamashita K, Ishitani R, Nureki O - Nat Commun (2015)

Structure and function of SemiSWEET.(a) Time course of [14C]-sucrose uptake by proteoliposomes containing EcSemiSWEET (solid black squares) or empty control liposomes (open black squares) (mean± s.e.m., n=6). (b) Plots of the sucrose uptake rate versus the extra-liposomal sucrose concentration (mean±s.e.m., n=3). (c) Overall structure of the inward-open SemiSWEET dimer, viewed parallel to the membrane (upper) or from the intracellular side (lower). In the ribbon representations (left), TM1, TM2, and TM3 of one protomer are coloured blue, green and red, respectively, and the other protomer is coloured grey. In the surface representations (right), the dimer is coloured according to the surface electrostatic potential. The cut-away surface is shown in the parallel view. The twofold axis is indicated by dashed lines and an almond-shaped symbol. (d) Overall structure of the outward-open SemiSWEET dimer, viewed parallel to the membrane (upper) and from the extracellular side (lower), coloured as in c.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4309421&req=5

f1: Structure and function of SemiSWEET.(a) Time course of [14C]-sucrose uptake by proteoliposomes containing EcSemiSWEET (solid black squares) or empty control liposomes (open black squares) (mean± s.e.m., n=6). (b) Plots of the sucrose uptake rate versus the extra-liposomal sucrose concentration (mean±s.e.m., n=3). (c) Overall structure of the inward-open SemiSWEET dimer, viewed parallel to the membrane (upper) or from the intracellular side (lower). In the ribbon representations (left), TM1, TM2, and TM3 of one protomer are coloured blue, green and red, respectively, and the other protomer is coloured grey. In the surface representations (right), the dimer is coloured according to the surface electrostatic potential. The cut-away surface is shown in the parallel view. The twofold axis is indicated by dashed lines and an almond-shaped symbol. (d) Overall structure of the outward-open SemiSWEET dimer, viewed parallel to the membrane (upper) and from the extracellular side (lower), coloured as in c.
Mentions: We screened the bacterial SemiSWEETs and identified E. coli SemiSWEET (EcSemiSWEET) as a suitable candidate for structural studies. EcSemiSWEET possesses the conserved Pro-Gln motif and shares 36% sequence identity and 57% similarity with Bradyrhizobium japonicum SemiSWEET, which was previously characterized as a sucrose uniporter (Supplementary Fig. 1)24. To test the sucrose transport activity of EcSemiSWEET, we reconstituted the purified protein into liposomes and measured [14C]-sucrose uptake into the proteoliposomes. The proteoliposomes containing EcSemiSWEET showed slow but significant [14C]-sucrose uptake, as compared with the control empty liposomes (Fig. 1a). The unusually slow uptake of EcSemiSWEET compared with those of eukaryotic SWEET transporters suggests that sucrose might not be the physiological substrate. Nonetheless, our transport assay confirmed the sucrose transport activity of EcSemiSWEET. The rate of [14C]-sucrose uptake increased linearly according to the extraliposomal sucrose concentration and was not saturated even up to 300 mM, indicating the low-affinity binding of EcSemiSWEET to sucrose (Fig. 1b). Previous studies also reported the low affinities of the plant SWEETs for sugars1314, suggesting a common transport mechanism.

Bottom Line: A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer.The conserved PQ-loop motif serves as a molecular hinge that enables the 'binder clip-like' motion of SemiSWEET.The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

ABSTRACT
SWEET family proteins mediate sugar transport across biological membranes and play crucial roles in plants and animals. The SWEETs and their bacterial homologues, the SemiSWEETs, are related to the PQ-loop family, which is characterized by highly conserved proline and glutamine residues (PQ-loop motif). Although the structures of the bacterial SemiSWEETs were recently reported, the conformational transition and the significance of the conserved motif in the transport cycle have remained elusive. Here we report crystal structures of SemiSWEET from Escherichia coli, in the both inward-open and outward-open states. A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer. The conserved PQ-loop motif serves as a molecular hinge that enables the 'binder clip-like' motion of SemiSWEET. The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

Show MeSH
Related in: MedlinePlus