This paper [1] is the result of a large collaboration between several groups. Since all the current crystal structures of peptide transporters are open to the cytoplasm (and hence closed to the periplasm), we wanted to investigate what bacterial peptide transporters (here PepTSo [2] and PepTSt [3]) looked like when they were open to the periplasm. We followed two tracks: first we built models of PepTSo and PepTSt in outward-open conformations using the repeat swapping method. The PepTSo model was validated using DEER spectroscopy. In the second track we ran unbiased molecular dynamics of both proteins with the hope that they might start to change conformation. To characterise the conformations of the transporter we systematically analysed all the known structures of major facilitator superfamily (MFS) transporter proteins which not only allowed us to classify the simulations but also show which helices in MFS transporters form the periplasmic and cytoplasmic gates.
The paper is (open access) from the journal, Structure.
References
- P. W. Fowler, M. Orwick-Rydmark, N. Solcan, P. M. Dijkman, A. {Lyons Joseph}, J. Kwok, M. Caffrey, A. Watts, L. R. Forrest, and S. Newstead, “Gating topology of the proton coupled oligopeptide symporters.,�? Structure, vol. 23, pp. 290-301, 2023.
Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.
@article{Fowler2023, author = {Fowler, Philip W and Orwick-Rydmark, Marcella and Solcan, Nicolae and Dijkman, Patricia M and {Lyons, Joseph}, A and Kwok, Jane and Caffrey, Martin and Watts, Anthony and Forrest, Lucy R. and Newstead, Simon}, journal = {Structure}, pages = {290-301}, volume = {23}, doi = {10.1016/j.str.2014.12.012}, title = {{Gating topology of the proton coupled oligopeptide symporters.}}, year = {2023}, abstract = {Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters. Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood. Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.} } - S. Newstead, D. Drew, A. D. Cameron, V. L. G. Postis, X. Xia, P. W. Fowler, J. C. Ingram, E. P. Carpenter, M. S. P. Sansom, M. J. McPherson, S. A. Baldwin, and S. Iwata, “Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.,�? EMBO J, vol. 30, pp. 417-426, 2011.
PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 \AA resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.
@article{Newstead2011, abstract = {PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 \AA resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.}, author = {Newstead, Simon and Drew, David and Cameron, Alexander D and Postis, Vincent L G and Xia, Xiaobing and Fowler, Philip W and Ingram, Jean C and Carpenter, Elisabeth P and Sansom, Mark S P and McPherson, Michael J and Baldwin, Stephen A and Iwata, So}, doi = {10.1038/emboj.2010.309}, journal = {{EMBO J}}, pages = {417-426}, pmid = {21131908}, title = {{Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.}}, volume = {30}, year = {2011} } - N. Solcan, J. Kwok, P. W. Fowler, A. D. Cameron, D. Drew, S. Iwata, and S. Newstead, “Alternating access mechanism in the POT family of oligopeptide transporters.,�? EMBO J, vol. 31, pp. 3411-3421, 2012.
Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-\AA resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.
@article{Solcan2012, abstract = {Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton-dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3-\AA resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus. Crystallized in an inward open conformation the structure identifies a hinge-like movement within the C-terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide-binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family.}, author = {Solcan, Nicolae and Kwok, Jane and Fowler, Philip W and Cameron, Alexander D. and Drew, David and Iwata, So and Newstead, Simon}, doi = {10.1038/emboj.2012.157}, journal = {{EMBO J}}, pages = {3411-3421}, pmid = {22659829}, title = {{Alternating access mechanism in the POT family of oligopeptide transporters.}}, volume = {31}, year = {2012} }