Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP
Abstract The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-44327-3 |
| _version_ | 1797355672582488064 |
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| author | Martin F. Peter Jan A. Ruland Yeojin Kim Philipp Hendricks Niels Schneberger Jan Peter Siebrasse Gavin H. Thomas Ulrich Kubitscheck Gregor Hagelueken |
| author_facet | Martin F. Peter Jan A. Ruland Yeojin Kim Philipp Hendricks Niels Schneberger Jan Peter Siebrasse Gavin H. Thomas Ulrich Kubitscheck Gregor Hagelueken |
| author_sort | Martin F. Peter |
| collection | DOAJ |
| description | Abstract The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa. |
| first_indexed | 2024-03-08T14:14:29Z |
| format | Article |
| id | doaj.art-b83022bdcbc940eaaa4826707fa4d729 |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-08T14:14:29Z |
| publishDate | 2024-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-b83022bdcbc940eaaa4826707fa4d7292024-01-14T12:28:45ZengNature PortfolioNature Communications2041-17232024-01-0115111210.1038/s41467-023-44327-3Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaPMartin F. Peter0Jan A. Ruland1Yeojin Kim2Philipp Hendricks3Niels Schneberger4Jan Peter Siebrasse5Gavin H. Thomas6Ulrich Kubitscheck7Gregor Hagelueken8Institute of Structural Biology, University of Bonn, Venusberg-Campus 1Clausius Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12Institute of Structural Biology, University of Bonn, Venusberg-Campus 1Institute of Structural Biology, University of Bonn, Venusberg-Campus 1Institute of Structural Biology, University of Bonn, Venusberg-Campus 1Clausius Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12Department of Biology (Area 10), University of YorkClausius Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12Institute of Structural Biology, University of Bonn, Venusberg-Campus 1Abstract The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.https://doi.org/10.1038/s41467-023-44327-3 |
| spellingShingle | Martin F. Peter Jan A. Ruland Yeojin Kim Philipp Hendricks Niels Schneberger Jan Peter Siebrasse Gavin H. Thomas Ulrich Kubitscheck Gregor Hagelueken Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP Nature Communications |
| title | Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP |
| title_full | Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP |
| title_fullStr | Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP |
| title_full_unstemmed | Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP |
| title_short | Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP |
| title_sort | conformational coupling of the sialic acid trap transporter hisiaqm with its substrate binding protein hisiap |
| url | https://doi.org/10.1038/s41467-023-44327-3 |
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