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}
  }

We recently examined how Kir1.1, an inwardly-rectifying potassium channel that is found in the kidneys, opens and closes in response to being stimulated by changes in pH or the presence of absence of PIP2, a signalling lipid [1]. The key result of that paper was that we could identify several networks of residues that came together to form one large gate when the channel was open. In this addendum paper, we examine how mutating several of these residues affected the kinetics of gating [2]. By comparing the on- and off-rates we are able to infer that the transition state more closely resembles that pre-open, rather than open, state. This paper is open access and is freely available to download.

References

@article{Bollepalli2014,
abstract = {X-ray crystallography has provided tremendous insight into the different structural states of membrane proteins and, in particular, of ion channels. However, the molecular forces that determine the thermodynamic stability of a particular state are poorly understood. Here we analyze the different X-ray structures of an inwardly rectifying potassium channel (Kir1.1) in relation to functional data we obtained for over 190 mutants in Kir1.1. This mutagenic perturbation analysis uncovered an extensive, state-dependent network of physically interacting residues that stabilizes the pre-open and open states of the channel, but fragments upon channel closure. We demonstrate that this gating network is an important structural determinant of the thermodynamic stability of these different gating states and determines the impact of individual mutations on channel function. These results have important implications for our understanding of not only K+ channel gating but also the more general nature of conformational transitions that occur in other allosteric proteins.},
author = {Bollepalli, Murali K. and Fowler, Philip W. and Rapedius, Markus and Shang, Lijun and Sansom, Mark S P and Tucker, Stephen J. and Baukrowitz, Thomas},
doi = {10.1016/j.str.2014.04.018},
journal = {Structure},
pages = {1037-1046},
pmid = {24980796},
title = {{State-dependent network connectivity determines gating in a K+ channel.}},
volume = {22},
year = {2014}
}

@article{Fowler2014,
abstract = {In a previous study we identified an extensive gating network within the inwardly rectifying Kir1.1 (ROMK) channel by combining systematic scanning mutagenesis and functional analysis with structural models of the channel in the closed, pre-open and open states. This extensive network appeared to stabilize the open and pre-open states, but the network fragmented upon channel closure. In this study we have analyzed the gating kinetics of different mutations within key parts of this gating network. These results suggest that the structure of the transition state (TS), which connects the pre-open and closed states of the channel, more closely resembles the structure of the pre-open state. Furthermore, the G-loop, which occurs at the centre of this extensive gating network, appears to become unstructured in the TS because mutations within this region have a ‘catalytic’ effect upon the channel gating kinetics.},
author = {Fowler, Philip W and Bollepalli, Murali K. and Rapedius, Markus and Nematian, Ehsan and Shang, Lijun and Sansom, Mark S. P. and Tucker, Stephen J. and Baukrowitz, Thomas},
doi = {10.4161/19336950.2014.962371},
journal = {Channels},
pages = {551-555},
title = {{Insights into the structural nature of the transition state in the Kir channel gating pathway}},
volume = {8},
year = {2014}
}

In we showed that the closed state of Kir1.1, a important potassium ion channel found in the kidneys, was stabilised by a single hydrogen bond. builds on that work by looking for any interactions that stabilise either the open or closed state of the channel by systematically mutating the majority of the residues to alanine. We were surprised to find that 47 mutations destabilised the open state but only 2 destabilised the closed state, one of which was the one we’d found before. Modelling suggests that this is because open conformations of the channel are more optimised and compact hence mutations tend to be more disruptive. The work was partly funded by the Wellcome Trust and hence is free to download.

In we examine how the , LacY, changes its structure to shuttle molecules of lactose across a cell membrane. The change in conformation is modelled usinga biased computational method called (DIMS) and the results compared to the results of some previously published . We conclude that LacY, as expected, does pass through an occluded intermediate and this is incompatible a simple rigid-body motion as implied by a “rocker-switch�? mechanism.

It is published in the and is  (open access).

Can we predict the conductance of a potassium ion channel from an experimental structure?

In  we examine the kinetic barriers experienced by potassium ions (and waters) as they move through the narrowest part of two different potassium ion channels. We examine the reproducibility of our results and test the sensitivity of the approach to changes in the method. We conclude that we are currently unable to accurately calculate the kinetic barriers to conduction for potassium channels and that other channels (such as sodium channels) may be more amenable to this approach.

This article is published in the and is (open access). It carries out the preparatory work necessary for on how potassium ions and water molecules move through the selectivity filter of a voltage-gated potassium ion channel.