# Publications

### 2015

• T. Reddy, D. Shorthouse, D. L. Parton, E. Jefferys, P. W. Fowler, M. Chavent, M. Baaden, and M. S. P. Sansom, “Nothing to sneeze at: a dynamic and integrative computational model of an influenza A virion,” Structure, vol. accepted, 2015.
@article{Reddy2015,
author = {Reddy, Tyler and Shorthouse, David and Parton, Daniel L and Jefferys, Elizabeth and Fowler, Philip W and Chavent, Matthieu and Baaden, Marc and Sansom, Mark S P},
journal = {Structure},
title = {{Nothing to sneeze at: a dynamic and integrative computational model of an influenza A virion}},
volume = {accepted},
year = {2015}
}

• 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, 2015.

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{Fowler2015,
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 = {2015},
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.}
}

### 2014

• P. W. Fowler, M. K. Bollepalli, M. Rapedius, E. Nematian, L. Shang, M. S. P. Sansom, S. J. Tucker, and T. Baukrowitz, “Insights into the structural nature of the transition state in the Kir channel gating pathway,” Channels, vol. 8, pp. 551-555, 2014.

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.

@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}
}

• E. Jefferys, M. S. P. Sansom, and P. W. Fowler, “NRas slows the rate at which a model lipid bilayer phase separates,” Faraday Discussions, vol. 169, pp. 209-223, 2014.

The Ras family of small membrane-associated GTP-ases are important components in many different cell signalling cascades. They are thought to cluster on the cell membrane through association with cholesterol-rich nanodomains. This process remains poorly understood. Here we test the effect of adding multiple copies of NRas, one of the canonical Ras proteins, to a three-component lipid bilayer that rapidly undergoes spinodal decomposition (i.e. unmixing), thereby creating ordered and disordered phases. Coarse-grained molecular dynamics simulations of a large bilayer containing 6000 lipids, with and without protein, are compared. NRas preferentially localises to the interface between the domains and slows the rate at which the domains grow. We infer that this doubly-lipidated cell signalling protein is reducing the line tension between the ordered and disordered regions. This analysis is facilitated by our use of techniques borrowed from image-processing. The conclusions above are contingent upon several assumptions, including the use of a model lipid with doubly unsaturated tails and the limited structural data available for the C-terminus of NRas, which is where the lipid anchors are found.

@article{Jefferys2014,
abstract = {The Ras family of small membrane-associated GTP-ases are important components in many different cell signalling cascades. They are thought to cluster on the cell membrane through association with cholesterol-rich nanodomains. This process remains poorly understood. Here we test the effect of adding multiple copies of NRas, one of the canonical Ras proteins, to a three-component lipid bilayer that rapidly undergoes spinodal decomposition (i.e. unmixing), thereby creating ordered and disordered phases. Coarse-grained molecular dynamics simulations of a large bilayer containing 6000 lipids, with and without protein, are compared. NRas preferentially localises to the interface between the domains and slows the rate at which the domains grow. We infer that this doubly-lipidated cell signalling protein is reducing the line tension between the ordered and disordered regions. This analysis is facilitated by our use of techniques borrowed from image-processing. The conclusions above are contingent upon several assumptions, including the use of a model lipid with doubly unsaturated tails and the limited structural data available for the C-terminus of NRas, which is where the lipid anchors are found.},
author = {Jefferys, Elizabeth and Sansom, Mark S. P. and Fowler, Philip W},
doi = {10.1039/c3fd00131h},
pages = {209-223},
title = {{NRas slows the rate at which a model lipid bilayer phase separates}},
volume = {169},
year = {2014}
}

• L. S. Stelzl, P. W. Fowler, M. S. P. Sansom, and O. Beckstein, “Flexible Gates Generate Occluded Intermediates in the Transport Cycle of LacY,” J Mol Biol, vol. 426, pp. 735-751, 2014.

The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepTSo, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates.

@article{Stelzl2013,
abstract = {The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepTSo, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates.},
author = {Stelzl, Lukas S. and Fowler, Philip W and Sansom, Mark S.P. and Beckstein, Oliver},
doi = {10.1016/j.jmb.2013.10.024},
journal = {{J Mol Biol}},
pages = {735-751},
pmid = {24513108},
title = {{Flexible Gates Generate Occluded Intermediates in the Transport Cycle of LacY}},
volume = {426},
year = {2014}
}

• M. K. Bollepalli, P. W. Fowler, M. Rapedius, L. Shang, M. S. P. Sansom, S. J. Tucker, and T. Baukrowitz, “State-dependent network connectivity determines gating in a K+ channel.,” Structure, vol. 22, pp. 1037-1046, 2014.

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.

@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}
}

### 2013

• P. W. Fowler, O. Beckstein, E. Abad, and M. S. P. Sansom, “Detailed Examination of a Single Conduction Event in a Potassium Channel,” J Phys Chem Lett, vol. 4, pp. 3104-3109, 2013.

Although extensively studied, it has proved difficult to describe in detail how potassium ion channels conduct cations and water. We present a computational study that, by using stratified umbrella sampling, examines nearly an entire conduction event of the Kv1.2/2.1 paddle chimera and thereby identifies the expected stable configurations of ions and waters in the selectivity filter of the channel. We describe in detail the motions of the ions and waters during a conduction event, focusing on how waters and ions enter the filter, the rotation of water molecules inside the filter, and how potassium ions are coordinated as they move from a water to a protein environment. Finally, we analyze the small conformational changes undergone by the protein, showing that the stable configurations are most similar to the experimental crystal structure.

@article{Fowler2013c,
abstract = {Although extensively studied, it has proved difficult to describe in detail how potassium ion channels conduct cations and water. We present a computational study that, by using stratified umbrella sampling, examines nearly an entire conduction event of the Kv1.2/2.1 paddle chimera and thereby identifies the expected stable configurations of ions and waters in the selectivity filter of the channel. We describe in detail the motions of the ions and waters during a conduction event, focusing on how waters and ions enter the filter, the rotation of water molecules inside the filter, and how potassium ions are coordinated as they move from a water to a protein environment. Finally, we analyze the small conformational changes undergone by the protein, showing that the stable configurations are most similar to the experimental crystal structure.},
author = {Fowler, Philip William and Beckstein, Oliver and Abad, Enrique and Sansom, Mark S. P.},
doi = {10.1021/jz4014079},
journal = {{J Phys Chem Lett}},
pages = {3104-3109},
pmid = {24143269},
title = {{Detailed Examination of a Single Conduction Event in a Potassium Channel}},
volume = {4},
year = {2013}
}

• P. W. Fowler, E. Abad, O. Beckstein, and M. S. P. Sansom, “Energetics of multi-ion conduction pathways in potassium ion channels,” J Chem Theory Comput, vol. 9, pp. 5176-5189, 2013.

Potassium ion channels form pores in cell membranes, allowing potassium ions through while preventing the passage of sodium ions. Despite numerous high-resolution structures, it is not yet possible to relate their structure to their single molecule function other than at a qualitative level. Over the past decade, there has been a concerted effort using molecular dynamics to capture the thermodynamics and kinetics of conduction by calculating potentials of mean force (PMF). These can be used, in conjunction with the electro-diffusion theory, to predict the conductance of a specific ion channel. Here, we calculate seven independent PMFs, thereby studying the differences between two potassium ion channels, the effect of the CHARMM CMAP forcefield correction, and the sensitivity and reproducibility of the method. Thermodynamically stable ion–water configurations of the selectivity filter can be identified from all the free energy landscapes, but the heights of the kinetic barriers for potassium ions to move through the selectivity filter are, in nearly all cases, too high to predict conductances in line with experiment. This implies it is not currently feasible to predict the conductance of potassium ion channels, but other simpler channels may be more tractable.

@article{Fowler2013b,
abstract = {Potassium ion channels form pores in cell membranes, allowing potassium ions through while preventing the passage of sodium ions. Despite numerous high-resolution structures, it is not yet possible to relate their structure to their single molecule function other than at a qualitative level. Over the past decade, there has been a concerted effort using molecular dynamics to capture the thermodynamics and kinetics of conduction by calculating potentials of mean force (PMF). These can be used, in conjunction with the electro-diffusion theory, to predict the conductance of a specific ion channel. Here, we calculate seven independent PMFs, thereby studying the differences between two potassium ion channels, the effect of the CHARMM CMAP forcefield correction, and the sensitivity and reproducibility of the method. Thermodynamically stable ion–water configurations of the selectivity filter can be identified from all the free energy landscapes, but the heights of the kinetic barriers for potassium ions to move through the selectivity filter are, in nearly all cases, too high to predict conductances in line with experiment. This implies it is not currently feasible to predict the conductance of potassium ion channels, but other simpler channels may be more tractable.},
author = {Fowler, Philip William and Abad, Enrique and Beckstein, Oliver and Sansom, Mark S. P.},
doi = {10.1021/ct4005933},
journal = {{J Chem Theory Comput}},
pages = {5176-5189},
pmid = {24353479},
title = {{Energetics of multi-ion conduction pathways in potassium ion channels}},
volume = {9},
year = {2013}
}

• P. W. Fowler and M. S. P. Sansom, “The pore of voltage-gated potassium ion channels is strained when closed.,” Nature Comms, vol. 4, p. 1872, 2013.

Voltage-gated potassium channels form potassium-selective pores in cell membranes. They open or close in response to changes in the transmembrane potential and are essential for generating action potentials, and thus for the functioning of heart and brain. While a mechanism for how these channels close has been proposed, it is not clear what drives their opening. Here we use free energy molecular dynamics simulations to show that work must be done on the pore to reduce the kink in the pore-lining (S6) $\alpha$-helices, thereby forming the helix bundle crossing and closing the channel. Strain is built up as the pore closes, which subsequently drives opening. We also determine the effect of mutating the PVPV motif that causes the kink in the S6 helix. Finally, an approximate upper limit on how far the S4 helix is displaced as the pore closes is estimated.

@article{Fowler2013,
abstract = {Voltage-gated potassium channels form potassium-selective pores in cell membranes. They open or close in response to changes in the transmembrane potential and are essential for generating action potentials, and thus for the functioning of heart and brain. While a mechanism for how these channels close has been proposed, it is not clear what drives their opening. Here we use free energy molecular dynamics simulations to show that work must be done on the pore to reduce the kink in the pore-lining (S6) $\alpha$-helices, thereby forming the helix bundle crossing and closing the channel. Strain is built up as the pore closes, which subsequently drives opening. We also determine the effect of mutating the PVPV motif that causes the kink in the S6 helix. Finally, an approximate upper limit on how far the S4 helix is displaced as the pore closes is estimated.},
author = {Fowler, Philip W and Sansom, Mark S. P.},
doi = {10.1038/ncomms2858},
journal = {Nature {C}omms},
pages = {1872},
pmid = {23695666},
title = {{The pore of voltage-gated potassium ion channels is strained when closed.}},
volume = {4},
year = {2013}
}

### 2012

• 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}
}

### 2011

• 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}
}

### 2010

• J. J. Paynter, I. Andres-Enguix, P. W. Fowler, S. Tottey, W. Cheng, D. Enkvetchakul, V. N. Bavro, Y. Kusakabe, M. S. P. Sansom, N. J. Robinson, C. G. Nichols, and S. J. Tucker, “Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.,” J Biol Chem, vol. 285, pp. 40754-40761, 2010.

The superfamily of prokaryotic inwardly-rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. However, relatively little is known about their regulation, or about their physiological role in vivo. In this study we have used random mutagenesis and genetic complementation in K+-auxotrophic E.coli and S.cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide-helix, transmembrane helices and C-terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favour a model of KirBac channel gating in which opening of the pore at the helix bundle crossing plays a far more important role than has recently been proposed.

@article{Paynter2010b,
abstract = {The superfamily of prokaryotic inwardly-rectifying (KirBac) potassium channels are homologous to mammalian Kir channels. However, relatively little is known about their regulation, or about their physiological role in vivo. In this study we have used random mutagenesis and genetic complementation in K+-auxotrophic E.coli and S.cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide-helix, transmembrane helices and C-terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favour a model of KirBac channel gating in which opening of the pore at the helix bundle crossing plays a far more important role than has recently been proposed.},
author = {Paynter, Jennifer J and Andres-Enguix, Isabelle and Fowler, Philip W and Tottey, Stephen and Cheng, Wayland and Enkvetchakul, Decha and Bavro, Vassiliy N and Kusakabe, Yoshio and Sansom, Mark S P and Robinson, Nigel J and Nichols, Colin G and Tucker, Stephen J},
doi = {10.1074/jbc.M110.175687},
journal = {{J Biol Chem}},
pages = {40754-40761},
pmid = {20876570},
title = {{Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains.}},
volume = {285},
year = {2010}
}

### 2009

• S. Newstead, P. W. Fowler, P. Bilton, E. P. Carpenter, P. J. Sadler, D. J. Campopiano, M. S. P. Sansom, and S. Iwata, “Insights into how nucleotide-binding domains power ABC transport.,” Structure, vol. 17, pp. 1213-1222, 2009.

The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered.

@article{Newstead2009,
abstract = {The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered.},
author = {Newstead, Simon and Fowler, Philip W and Bilton, Paul and Carpenter, Elisabeth P and Sadler, Peter J and Campopiano, Dominic J and Sansom, Mark S P and Iwata, So},
doi = {10.1016/j.str.2009.07.009},
journal = {Structure},
pages = {1213-1222},
pmid = {19748342},
title = {{Insights into how nucleotide-binding domains power ABC transport.}},
volume = {17},
year = {2009}
}

• E. Abad, J. Reingruber, P. Fowler, M. S. P. Sansom, D. Wei, and X. Wang, “A Novel Rate Theory Approach To Transport In Ion Channels,” AIP Conf Proc, vol. 1102, pp. 236-243, 2009.

We present a novel rate theory based on the notions of splitting probability and mean first passage time to describe conduction of single ions in narrow, effectively 1D membrane channels. In contrast to traditional approaches such as transition state theory or Kramers theory, transitions between different conduction states in our model are governed by rates which depend on the full geometry of the potential of mean force (PMF) resulting from the superposition of an equilibrium free energy profile and a transmembrane potential induced by a nonequilibrium constraint. If a detailed theoretical PMF is available (e.g. from atomistic molecular dynamics simulations), it can be used to compute characteristic conductance curves in the framework of our model, thereby bridging the gap between the atomistic and the mesoscopic level of description. Explicit analytic solutions for the rates, the ion flux and the associated electric current can be obtained by approximating the actual PMF by a piecewise linear potential.

@article{Abad2009,
abstract = {We present a novel rate theory based on the notions of splitting probability and mean first passage time to describe conduction of single ions in narrow, effectively 1D membrane channels. In contrast to traditional approaches such as transition state theory or Kramers theory, transitions between different conduction states in our model are governed by rates which depend on the full geometry of the potential of mean force (PMF) resulting from the superposition of an equilibrium free energy profile and a transmembrane potential induced by a nonequilibrium constraint. If a detailed theoretical PMF is available (e.g. from atomistic molecular dynamics simulations), it can be used to compute characteristic conductance curves in the framework of our model, thereby bridging the gap between the atomistic and the mesoscopic level of description. Explicit analytic solutions for the rates, the ion flux and the associated electric current can be obtained by approximating the actual PMF by a piecewise linear potential.},
author = {Abad, Enrique and Reingruber, J and Fowler, P. and Sansom, Mark S P and Wei, Dong-Qing and Wang, Xi-Jun},
doi = {10.1063/1.3108380},
editor = {Wei, Dong-Qing},
journal = {{AIP Conf Proc}},
pages = {236-243},
publisher = {AIP},
title = {{A Novel Rate Theory Approach To Transport In Ion Channels}},
volume = {1102},
year = {2009}
}

### 2008

• K. Tai, P. W. Fowler, Y. Mokrab, P. Stansfeld, and M. S. P. Sansom, “Molecular modeling and simulation studies of ion channel structures, dynamics and mechanisms.,” in Methods in cell nano biology, First ed., B. P. Jena, Ed., Elsevier B.V., 2008, vol. 90, pp. 233-265.

Ion channels are integral membrane proteins that enable selected ions to flow passively across membranes. Channel proteins have been the focus of computational approaches to relate their three-dimensional (3D) structure to their physiological function. We describe a number of computational tools to model ion channels. Homology modeling may be used to construct structural models of channels based on available X-ray structures. Electrostatics calculations enable an approximate evaluation of the energy profile of an ion passing through a channel. Molecular dynamics simulations and free-energy calculations provide information on the thermodynamics and kinetics of channel function.

@incollection{Tai2008,
abstract = {Ion channels are integral membrane proteins that enable selected ions to flow passively across membranes. Channel proteins have been the focus of computational approaches to relate their three-dimensional (3D) structure to their physiological function. We describe a number of computational tools to model ion channels. Homology modeling may be used to construct structural models of channels based on available X-ray structures. Electrostatics calculations enable an approximate evaluation of the energy profile of an ion passing through a channel. Molecular dynamics simulations and free-energy calculations provide information on the thermodynamics and kinetics of channel function.},
author = {Tai, Kaihsu and Fowler, Philip W and Mokrab, Younes and Stansfeld, Phillip and Sansom, Mark S P},
booktitle = {Methods in Cell Nano Biology},
chapter = {12},
doi = {10.1016/S0091-679X(08)00812-1},
edition = {First},
editor = {Jena, Bhanu P},
issn = {0091-679X},
pages = {233-265},
pmid = {19195554},
publisher = {Elsevier B.V.},
title = {{Molecular modeling and simulation studies of ion channel structures, dynamics and mechanisms.}},
volume = {90},
year = {2008}
}

• P. W. Fowler, K. Tai, and M. S. P. Sansom, “The selectivity of K+ ion channels: testing the hypotheses.,” Biophys J, vol. 95, pp. 5062-5072, 2008.

How K(+) channels are able to conduct certain cations yet not others remains an important but unresolved question. The recent elucidation of the structure of NaK, an ion channel that conducts both Na(+) and K(+) ions, offers an opportunity to test the various hypotheses that have been put forward to explain the selectivity of K(+) ion channels. We test the snug-fit, field-strength, and over-coordination hypotheses by comparing their predictions to the results of classical molecular dynamics simulations of the K(+) selective channel KcsA and the less selective channel NaK embedded in lipid bilayers. Our results are incompatible with the so-called strong variant of the snug-fit hypothesis but are consistent with the over-coordination hypothesis and neither confirm nor refute the field-strength hypothesis. We also find that the ions and waters in the NaK selectivity filter unexpectedly move to a new conformation in seven K(+) simulations: the two K(+) ions rapidly move from site S4 to S2 and from the cavity to S4. At the same time, the selectivity filter narrows around sites S1 and S2 and the carbonyl oxygen atoms rotate 20 degrees -40 degrees inwards toward the ion. These motions diminish the large structural differences between the crystallographic structures of the selectivity filters of NaK and KcsA and appear to allow the binding of ions to S2 of NaK at physiological temperature.

@article{Fowler2008,
abstract = {How K(+) channels are able to conduct certain cations yet not others remains an important but unresolved question. The recent elucidation of the structure of NaK, an ion channel that conducts both Na(+) and K(+) ions, offers an opportunity to test the various hypotheses that have been put forward to explain the selectivity of K(+) ion channels. We test the snug-fit, field-strength, and over-coordination hypotheses by comparing their predictions to the results of classical molecular dynamics simulations of the K(+) selective channel KcsA and the less selective channel NaK embedded in lipid bilayers. Our results are incompatible with the so-called strong variant of the snug-fit hypothesis but are consistent with the over-coordination hypothesis and neither confirm nor refute the field-strength hypothesis. We also find that the ions and waters in the NaK selectivity filter unexpectedly move to a new conformation in seven K(+) simulations: the two K(+) ions rapidly move from site S4 to S2 and from the cavity to S4. At the same time, the selectivity filter narrows around sites S1 and S2 and the carbonyl oxygen atoms rotate 20 degrees -40 degrees inwards toward the ion. These motions diminish the large structural differences between the crystallographic structures of the selectivity filters of NaK and KcsA and appear to allow the binding of ions to S2 of NaK at physiological temperature.},
author = {Fowler, Philip W and Tai, Kaihsu and Sansom, Mark S P},
doi = {10.1529/biophysj.108.132035},
journal = {{Biophys J}},
pages = {5062-5072},
pmid = {18790851},
title = {{The selectivity of K+ ion channels: testing the hypotheses.}},
volume = {95},
year = {2008}
}

• E. Psachoulia, P. W. Fowler, P. J. Bond, and M. S. P. Sansom, “Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin A helix dimerization.,” Biochemistry, vol. 47, pp. 10503-10512, 2008.

Oligomerization of transmembrane (TM) helices is a key stage in the folding of membrane proteins. Glycophorin A (GpA) is a well-documented test system for this process. Coarse-grained molecular dynamics (CG-MD) allows us to simulate the self-assembly of TM helices into dimers, for both wild-type (WT) and mutant GpA sequences. For the WT sequences, dimers formed rapidly and remained stable in all simulations. The resultant dimers exhibited right-handed crossing and the same interhelix contacts as in NMR structures. Simulations of disruptive mutants revealed the dimers were less stable, with values of DeltaDelta G dimerization consistent with experimental data. The dimers of disruptive mutants were distorted relative to the WT and showed left-handed crossing of their helices. CG-MD can therefore be used to explore the interactions of TM helices, an important stage in the folding of membrane proteins. In particular, CG-MD has been shown to be sensitive enough to detect disruptions introduced by mutation. Future refinement of such models via atomistic simulations will enable a multiscale approach to predict the folding of membrane proteins.

@article{Psachoulia2008,
abstract = {Oligomerization of transmembrane (TM) helices is a key stage in the folding of membrane proteins. Glycophorin A (GpA) is a well-documented test system for this process. Coarse-grained molecular dynamics (CG-MD) allows us to simulate the self-assembly of TM helices into dimers, for both wild-type (WT) and mutant GpA sequences. For the WT sequences, dimers formed rapidly and remained stable in all simulations. The resultant dimers exhibited right-handed crossing and the same interhelix contacts as in NMR structures. Simulations of disruptive mutants revealed the dimers were less stable, with values of DeltaDelta G dimerization consistent with experimental data. The dimers of disruptive mutants were distorted relative to the WT and showed left-handed crossing of their helices. CG-MD can therefore be used to explore the interactions of TM helices, an important stage in the folding of membrane proteins. In particular, CG-MD has been shown to be sensitive enough to detect disruptions introduced by mutation. Future refinement of such models via atomistic simulations will enable a multiscale approach to predict the folding of membrane proteins.},
author = {Psachoulia, Emi and Fowler, Philip W and Bond, Peter J and Sansom, Mark S P},
doi = {10.1021/bi800678t},
journal = {Biochemistry},
pages = {10503-10512},
pmid = {18783247},
title = {{Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin A helix dimerization.}},
volume = {47},
year = {2008}
}

### 2007

• P. W. Fowler, S. Geroult, S. Jha, G. Waksman, and P. V. Coveney, “Rapid, accurate, and precise calculation of relative binding affinities for the SH2 domain using a computational grid,” J Chem Theory Comput, vol. 3, pp. 1193-1202, 2007.

We describe and apply a method that reduces the time taken to calculate binding free energies using thermodynamic integration. This method uses a stack of grid software, which we call STIMD, that allows the scientist to easily distribute the necessary simulations around a computational grid thereby accelerating the process. We use this method to study how a series of phosphopeptides binds to the Src SH2 domain. The binding of phosphopeptides to the Src SH2 domain is described by the “two-pronged plug two-holed socket” model, and we investigate this model by reducing the length of the aliphatic side chain that engages the second of the two sockets through two successive alchemical mutations. Seven different values of $\Delta$$\DeltaG have been calculated, and we report good agreement with experiment. We then propose an extension to this model using the insights gained from a free energy component analysis. @article{Fowler2007a, abstract = {We describe and apply a method that reduces the time taken to calculate binding free energies using thermodynamic integration. This method uses a stack of grid software, which we call STIMD, that allows the scientist to easily distribute the necessary simulations around a computational grid thereby accelerating the process. We use this method to study how a series of phosphopeptides binds to the Src SH2 domain. The binding of phosphopeptides to the Src SH2 domain is described by the “two-pronged plug two-holed socket” model, and we investigate this model by reducing the length of the aliphatic side chain that engages the second of the two sockets through two successive alchemical mutations. Seven different values of \Delta$$\Delta$G have been calculated, and we report good agreement with experiment. We then propose an extension to this model using the insights gained from a free energy component analysis.},
author = {Fowler, Philip W and Geroult, Sebastien and Jha, Shantenu and Waksman, Gabriel and Coveney, Peter V.},
doi = {10.1021/ct6003017},
journal = {{J Chem Theory Comput}},
pages = {1193-1202},
title = {{Rapid, accurate, and precise calculation of relative binding affinities for the SH2 domain using a computational grid}},
volume = {3},
year = {2007}
}

• P. W. Fowler, K. Balali-Mood, S. Deol, P. V. Coveney, and M. S. P. Sansom, “Monotopic enzymes and lipid bilayers: a comparative study.,” Biochemistry, vol. 46, pp. 3108-3115, 2007.

Monotopic proteins make up a class of membrane proteins that bind tightly to, but do not span, cell membranes. We examine and compare how two monotopic proteins, monoamine oxidase B (MAO-B) and cyclooxygenase-2 (COX-2), interact with a phospholipid bilayer using molecular dynamics simulations. Both enzymes form between three and seven hydrogen bonds with the bilayer in our simulations with basic side chains accounting for the majority of these interactions. By analyzing lipid order parameters, we show that, to a first approximation, COX-2 disrupts only the upper leaflet of the bilayer. In contrast, the top and bottom halves of the lipid tails surrounding MAO-B are more and less ordered, respectively, than in the absence of the protein. Finally, we identify which residues are important in binding individual phospholipids by counting the number and type of lipid atoms that come close to each amino acid residue. The existing models that explain how these proteins bind to bilayers were proposed following inspection of the X-ray crystallographic structures. Our results support these models and suggest that basic residues contribute significantly to the binding of these monotopic proteins to bilayers through the formation of hydrogen bonds with phospholipids.

@article{Fowler2007,
abstract = {Monotopic proteins make up a class of membrane proteins that bind tightly to, but do not span, cell membranes. We examine and compare how two monotopic proteins, monoamine oxidase B (MAO-B) and cyclooxygenase-2 (COX-2), interact with a phospholipid bilayer using molecular dynamics simulations. Both enzymes form between three and seven hydrogen bonds with the bilayer in our simulations with basic side chains accounting for the majority of these interactions. By analyzing lipid order parameters, we show that, to a first approximation, COX-2 disrupts only the upper leaflet of the bilayer. In contrast, the top and bottom halves of the lipid tails surrounding MAO-B are more and less ordered, respectively, than in the absence of the protein. Finally, we identify which residues are important in binding individual phospholipids by counting the number and type of lipid atoms that come close to each amino acid residue. The existing models that explain how these proteins bind to bilayers were proposed following inspection of the X-ray crystallographic structures. Our results support these models and suggest that basic residues contribute significantly to the binding of these monotopic proteins to bilayers through the formation of hydrogen bonds with phospholipids.},
author = {Fowler, Philip W and Balali-Mood, Kia and Deol, Sundeep and Coveney, Peter V and Sansom, Mark S P},
doi = {10.1021/bi602455n},
journal = {Biochemistry},
pages = {3108-3115},
pmid = {17311421},
title = {{Monotopic enzymes and lipid bilayers: a comparative study.}},
volume = {46},
year = {2007}
}

• M. Rapedius, P. W. Fowler, L. Shang, M. S. P. Sansom, S. J. Tucker, and T. Baukrowitz, “H bonding at the helix-bundle crossing controls gating in Kir potassium channels.,” Neuron, vol. 55, pp. 602-614, 2007.

Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.

@article{Rapedius2007,
abstract = {Specific stimuli such as intracellular H+ and phosphoinositides (e.g., PIP2) gate inwardly rectifying potassium (Kir) channels by controlling the reversible transition between the closed and open states. This gating mechanism underlies many aspects of Kir channel physiology and pathophysiology; however, its structural basis is not well understood. Here, we demonstrate that H+ and PIP2 use a conserved gating mechanism defined by similar structural changes in the transmembrane (TM) helices and the selectivity filter. Our data support a model in which the gating motion of the TM helices is controlled by an intrasubunit hydrogen bond between TM1 and TM2 at the helix-bundle crossing, and we show that this defines a common gating motif in the Kir channel superfamily. Furthermore, we show that this proposed H-bonding interaction determines Kir channel pH sensitivity, pH and PIP2 gating kinetics, as well as a K+-dependent inactivation process at the selectivity filter and therefore many of the key regulatory mechanisms of Kir channel physiology.},
author = {Rapedius, Markus and Fowler, Philip W and Shang, Lijun and Sansom, Mark S P and Tucker, Stephen J and Baukrowitz, Thomas},
doi = {10.1016/j.neuron.2007.07.026},
journal = {Neuron},
pages = {602-614},
pmid = {17698013},
title = {{H bonding at the helix-bundle crossing controls gating in Kir potassium channels.}},
volume = {55},
year = {2007}
}

• M. Rapedius, J. J. Paynter, P. W. Fowler, L. Shang, M. S. P. Sansom, S. J. Tucker, and T. Baukrowitz, “Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing.,” Channels, vol. 1, pp. 327-330, 2007.

Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.

@article{Rapedius2007a,
abstract = {Inhibition by intracellular H(+) (pH gating) and activation by phosphoinositides such as PIP(2) (PIP(2)-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP(2) gating and pH gating are controlled by an intra-subunit H-bond at the helix-bundle crossing between a lysine in TM1 and a backbone carbonyl group in TM2. This interaction only occurs in the closed state and channel opening requires this H-bond to be broken, thereby influencing the kinetics of PIP(2) and pH gating in Kir channels. In this addendum, we explore the role of H-bonding in heteromeric Kir4.1/Kir5.1 channels. Kir5.1 subunits do not possess a TM1 lysine. However, homology modelling and molecular dynamics simulations demonstrate that the TM1 lysine in Kir4.1 is capable of H-bonding at the helix-bundle crossing. Consistent with this, the rates of pH and PIP2 gating in Kir4.1/Kir5.1 channels (two H-bonds) were intermediate between those of wild-type homomeric Kir4.1 (four H-bonds) and Kir4.1(K67M) channels (no H-bonds) suggesting that the number of H-bonds in the tetrameric channel complex determines the gating kinetics. Furthermore, in heteromeric Kir4.1(K67M)/Kir5.1 channels, where the two remaining H-bonds are disrupted, we found that the gating kinetics were similar to Kir4.1(K67M) homomeric channels despite the fact that these two channels differ considerably in their PIP(2) affinities. This indicates that Kir channel PIP(2) affinity has little impact on either the PIP(2) or pH gating kinetics.},
author = {Rapedius, Markus and Paynter, Jennifer J and Fowler, Philip W and Shang, Lijun and Sansom, Mark S P and Tucker, Stephen J and Baukrowitz, Thomas},
journal = {Channels},
pages = {327-330},
pmid = {18690035},
title = {{Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing.}},
volume = {1},
year = {2007}
}

### 2006

• P. W. Fowler, “Qualitative and Quantitative Aspects of Biomolecular Systems Revealed by Large Scale and Grid Computing Methods,” PhD Thesis, 2006.

Biomolecules, for example proteins, are dynamic entities and their interactions are important in determining the behaviour of cells. We shall use computational methods, specifically classical molecular dynamics, to simulate the dynamics of several biomolecular systems. The free energy of binding is the key quantitative measure of how strongly two molecules interact. Although theoretical methods exist for calculating these free energies, their use and validation has been hampered by the length of time it takes to compute a single value. We shall describe a novel method, steered thermodynamic integration using molecular dynamics (or STIMD), that uses a computational grid to accelerate from months to less than one week the time taken to calculate a single difference in binding free energy ($\Delta \Delta G$) and its use to study the binding of a peptide to a v-Src SH2 protein domain. Two related mutations are studied and the calculated and experimental values of $\Delta \Delta G$ are compared. We shall then discuss the insight into the binding event gained through the decomposition of the free energy by components of the system. The problems we encountered when using STIMD are described and several future developments are discussed. We shall discuss our protocol for integrating a prostaglandin H2 synthase (PGHS) monomer protein into a phospholipid bilayer and present evidence that the protein is correctly integrated. This is the first step in a qualitative study of the dynamical differences between the two isozymes of PGHS, the target for non-steroidal anti-inflammatory drugs, and we shall also describe several observed dynamical differences. We have used high-performance computing and computational grids for all these quantitative and qualitative studies as using classical molecular dynamics to simulate these biomolecular systems is extremely computationally intensive.

@phdthesis{Fowler2006a,
abstract = {Biomolecules, for example proteins, are dynamic entities and their interactions are important in determining the behaviour of cells. We shall use computational methods, specifically classical molecular dynamics, to simulate the dynamics of several biomolecular systems. The free energy of binding is the key quantitative measure of how strongly two molecules interact. Although theoretical methods exist for calculating these free energies, their use and validation has been hampered by the length of time it takes to compute a single value. We shall describe a novel method, steered thermodynamic integration using molecular dynamics (or STIMD), that uses a computational grid to accelerate from months to less than one week the time taken to calculate a single difference in binding free energy ($\Delta \Delta G$) and its use to study the binding of a peptide to a v-Src SH2 protein domain. Two related mutations are studied and the calculated and experimental values of $\Delta \Delta G$ are compared. We shall then discuss the insight into the binding event gained through the decomposition of the free energy by components of the system. The problems we encountered when using STIMD are described and several future developments are discussed. We shall discuss our protocol for integrating a prostaglandin H2 synthase (PGHS) monomer protein into a phospholipid bilayer and present evidence that the protein is correctly integrated. This is the first step in a qualitative study of the dynamical differences between the two isozymes of PGHS, the target for non-steroidal anti-inflammatory drugs, and we shall also describe several observed dynamical differences. We have used high-performance computing and computational grids for all these quantitative and qualitative studies as using classical molecular dynamics to simulate these biomolecular systems is extremely computationally intensive.},
author = {Fowler, Philip W},
school = {University College London},
title = {{Qualitative and Quantitative Aspects of Biomolecular Systems Revealed by Large Scale and Grid Computing Methods}},
year = {2006},
url = {https://impactstory.org/philipwfowler/product/97wswb006iva7m87zi05p4py}
}

• P. W. Fowler and P. V. Coveney, “A computational protocol for the integration of the monotopic protein prostaglandin H2 synthase into a phospholipid bilayer.,” Biophys J, vol. 91, pp. 401-410, 2006.

Prostaglandin H2 synthase (PGHS) synthesizes PGH2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.

@article{Fowler2006,
abstract = {Prostaglandin H2 synthase (PGHS) synthesizes PGH2, a prostaglandin precursor, from arachidonic acid and was the first monotopic enzyme to have its structure experimentally determined. Both isozymes of PGHS are inhibited by nonsteroidal antiinflammatory drugs, an important class of drugs that are the primary means of relieving pain and inflammation. Selectively inhibiting the second isozyme, PGHS-2, minimizes the gastrointestinal side-effects. This had been achieved by the new PGHS-2 selective NSAIDs (i.e., COX-2 inhibitors) but it has been recently suggested that they suffer from additional side-effects. The design of these drugs only made use of static structures from x-ray crystallographic experiments. Investigating the dynamics of both PGHS-1 and PGHS-2 using classical molecular dynamics is expected to generate new insight into the differences in behavior between the isozymes, and therefore may allow improved PGHS-2 selective inhibitors to be designed. We describe a molecular dynamics protocol that integrates PGHS monomers into phospholipid bilayers, thereby producing in silico atomistic models of the PGHS system. Our protocol exploits the vacuum created beneath the protein when several lipids are removed from the top leaflet of the bilayer. The protein integrates into the bilayer during the first 5 ns in a repeatable process. The integrated PGHS monomer is stable and forms multiple hydrogen bonds between the phosphate groups of the lipids and conserved basic residues (Arg, Lys) on the protein. These interactions stabilize the system and are similar to interactions observed for transmembrane proteins.},
author = {Fowler, Philip W and Coveney, Peter V},
doi = {10.1529/biophysj.105.077784},
journal = {{Biophys J}},
pages = {401-410},
pmid = {16632499},
title = {{A computational protocol for the integration of the monotopic protein prostaglandin H2 synthase into a phospholipid bilayer.}},
volume = {91},
year = {2006}
}

### 2005

• F. Giordanetto, P. W. Fowler, M. Saqi, and P. V. Coveney, “Large scale molecular dynamics simulation of native and mutant dihydropteroate synthase-sulphanilamide complexes suggests the molecular basis for dihydropteroate synthase drug resistance.,” Phil Trans R Soc Lond A, vol. 363, iss. 1833, pp. 2055-2073, 2005.

Antibiotic resistance is hampering the efficacy of drugs in the treatment of several pathological infections. Dihydropteroate synthase (DHPS) has been targeted by sulphonamide inhibitors for the past 60 years and has developed different amino acid mutations to survive sulpha drug action. We couple homology modelling techniques and massively parallel molecular dynamics simulations to study both the drug-bound and apo forms of native and mutant DHPS. Simulations of the complex between sulphanilamide and Streptomyces pneumoniae, DHPS shows how sulphanilamide is able to position itself close to 6-hydroxymethyl-7, 8-dihydropteridine-phosphate in a suitable position for the enzymatic transformation whereas in the mutant complex the sulpha drug is expelled from the catalytic site. Our simulations, therefore, provide insight into the molecular basis for drug resistance with S. pneumoniae DHPS.

@article{Giordanetto2005,
abstract = {Antibiotic resistance is hampering the efficacy of drugs in the treatment of several pathological infections. Dihydropteroate synthase (DHPS) has been targeted by sulphonamide inhibitors for the past 60 years and has developed different amino acid mutations to survive sulpha drug action. We couple homology modelling techniques and massively parallel molecular dynamics simulations to study both the drug-bound and apo forms of native and mutant DHPS. Simulations of the complex between sulphanilamide and Streptomyces pneumoniae, DHPS shows how sulphanilamide is able to position itself close to 6-hydroxymethyl-7, 8-dihydropteridine-phosphate in a suitable position for the enzymatic transformation whereas in the mutant complex the sulpha drug is expelled from the catalytic site. Our simulations, therefore, provide insight into the molecular basis for drug resistance with S. pneumoniae DHPS.},
author = {Giordanetto, Fabrizio and Fowler, Philip W and Saqi, Mansoor and Coveney, Peter V},
doi = {10.1098/rsta.2005.1629},
journal = {{Phil Trans R Soc Lond A}},
number = {1833},
pages = {2055-2073},
pmid = {16099766},
title = {{Large scale molecular dynamics simulation of native and mutant dihydropteroate synthase-sulphanilamide complexes suggests the molecular basis for dihydropteroate synthase drug resistance.}},
volume = {363},
year = {2005}
}

• P. W. Fowler, S. Jha, and P. V. Coveney, “Grid-based steered thermodynamic integration accelerates the calculation of binding free energies.,” Phil Trans R Soc Lond A, vol. 363, iss. 1833, pp. 1999-2015, 2005.

The calculation of binding free energies is important in many condensed matter problems. Although formally exact computational methods have the potential to complement, add to, and even compete with experimental approaches, they are difficult to use and extremely time consuming. We describe a Grid-based approach for the calculation of relative binding free energies, which we call Steered Thermodynamic Integration calculations using Molecular Dynamics (STIMD), and its application to Src homology 2 (SH2) protein cell signalling domains. We show that the time taken to compute free energy differences using thermodynamic integration can be significantly reduced: potentially from weeks or months to days of wall-clock time. To be able to perform such accelerated calculations requires the ability to both run concurrently and control in realtime several parallel simulations on a computational Grid. We describe how the RealityGrid computational steering system, in conjunction with a scalable classical MD code, can be used to dramatically reduce the time to achieve a result. This is necessary to improve the adoption of this technique and further allows more detailed investigations into the accuracy and precision of thermodynamic integration. Initial results for the Src SH2 system are presented and compared to a reported experimental value. Finally, we discuss the significance of our approach.

@article{Fowler2005,
abstract = {The calculation of binding free energies is important in many condensed matter problems. Although formally exact computational methods have the potential to complement, add to, and even compete with experimental approaches, they are difficult to use and extremely time consuming. We describe a Grid-based approach for the calculation of relative binding free energies, which we call Steered Thermodynamic Integration calculations using Molecular Dynamics (STIMD), and its application to Src homology 2 (SH2) protein cell signalling domains. We show that the time taken to compute free energy differences using thermodynamic integration can be significantly reduced: potentially from weeks or months to days of wall-clock time. To be able to perform such accelerated calculations requires the ability to both run concurrently and control in realtime several parallel simulations on a computational Grid. We describe how the RealityGrid computational steering system, in conjunction with a scalable classical MD code, can be used to dramatically reduce the time to achieve a result. This is necessary to improve the adoption of this technique and further allows more detailed investigations into the accuracy and precision of thermodynamic integration. Initial results for the Src SH2 system are presented and compared to a reported experimental value. Finally, we discuss the significance of our approach.},
author = {Fowler, Philip W and Jha, Shantenu and Coveney, Peter V},
doi = {10.1098/rsta.2005.1625},
journal = {{Phil Trans R Soc Lond A}},
number = {1833},
pages = {1999-2015},
pmid = {16099763},
title = {{Grid-based steered thermodynamic integration accelerates the calculation of binding free energies.}},
volume = {363},
year = {2005}
}

• P. V. Coveney and P. W. Fowler, “Modelling biological complexity: a physical scientist’s perspective.,” J R Soc Lond Interface, vol. 2, pp. 267-280, 2005.

We discuss the modern approaches of complexity and self-organization to understanding dynamical systems and how these concepts can inform current interest in systems biology. From the perspective of a physical scientist, it is especially interesting to examine how the differing weights given to philosophies of science in the physical and biological sciences impact the application of the study of complexity. We briefly describe how the dynamics of the heart and circadian rhythms, canonical examples of systems biology, are modelled by sets of nonlinear coupled differential equations, which have to be solved numerically. A major difficulty with this approach is that all the parameters within these equations are not usually known. Coupled models that include biomolecular detail could help solve this problem. Coupling models across large ranges of length- and time-scales is central to describing complex systems and therefore to biology. Such coupling may be performed in at least two different ways, which we refer to as hierarchical and hybrid multiscale modelling. While limited progress has been made in the former case, the latter is only beginning to be addressed systematically. These modelling methods are expected to bring numerous benefits to biology, for example, the properties of a system could be studied over a wider range of length- and time-scales, a key aim of systems biology. Multiscale models couple behaviour at the molecular biological level to that at the cellular level, thereby providing a route for calculating many unknown parameters as well as investigating the effects at, for example, the cellular level, of small changes at the biomolecular level, such as a genetic mutation or the presence of a drug. The modelling and simulation of biomolecular systems is itself very computationally intensive; we describe a recently developed hybrid continuum-molecular model, HybridMD, and its associated molecular insertion algorithm, which point the way towards the integration of molecular and more coarse-grained representations of matter. The scope of such integrative approaches to complex systems research is circumscribed by the computational resources available. Computational grids should provide a step jump in the scale of these resources; we describe the tools that RealityGrid, a major UK e-Science project, has developed together with our experience of deploying complex models on nascent grids. We also discuss the prospects for mathematical approaches to reducing the dimensionality of complex networks in the search for universal systems-level properties, illustrating our approach with a description of the origin of life according to the RNA world view.

@article{Coveney2005,
abstract = {We discuss the modern approaches of complexity and self-organization to understanding dynamical systems and how these concepts can inform current interest in systems biology. From the perspective of a physical scientist, it is especially interesting to examine how the differing weights given to philosophies of science in the physical and biological sciences impact the application of the study of complexity. We briefly describe how the dynamics of the heart and circadian rhythms, canonical examples of systems biology, are modelled by sets of nonlinear coupled differential equations, which have to be solved numerically. A major difficulty with this approach is that all the parameters within these equations are not usually known. Coupled models that include biomolecular detail could help solve this problem. Coupling models across large ranges of length- and time-scales is central to describing complex systems and therefore to biology. Such coupling may be performed in at least two different ways, which we refer to as hierarchical and hybrid multiscale modelling. While limited progress has been made in the former case, the latter is only beginning to be addressed systematically. These modelling methods are expected to bring numerous benefits to biology, for example, the properties of a system could be studied over a wider range of length- and time-scales, a key aim of systems biology. Multiscale models couple behaviour at the molecular biological level to that at the cellular level, thereby providing a route for calculating many unknown parameters as well as investigating the effects at, for example, the cellular level, of small changes at the biomolecular level, such as a genetic mutation or the presence of a drug. The modelling and simulation of biomolecular systems is itself very computationally intensive; we describe a recently developed hybrid continuum-molecular model, HybridMD, and its associated molecular insertion algorithm, which point the way towards the integration of molecular and more coarse-grained representations of matter. The scope of such integrative approaches to complex systems research is circumscribed by the computational resources available. Computational grids should provide a step jump in the scale of these resources; we describe the tools that RealityGrid, a major UK e-Science project, has developed together with our experience of deploying complex models on nascent grids. We also discuss the prospects for mathematical approaches to reducing the dimensionality of complex networks in the search for universal systems-level properties, illustrating our approach with a description of the origin of life according to the RNA world view.},
author = {Coveney, Peter V and Fowler, Philip W},
doi = {10.1098/rsif.2005.0045},
journal = {{J R Soc Lond Interface}},
pages = {267-280},
pmid = {16849185},
title = {{Modelling biological complexity: a physical scientist's perspective.}},
volume = {2},
year = {2005}
}

### 2004

• P. W. Fowler, P. V. Coveney, S. Jha, and S. Wan, “Exact calculation of peptide-protein binding energies by steered thermodynamic integration using high performance computing grids,” in Proceedings of the uk e-science all hands meeting, 2004.

We describe a grid-based method to dramatically accelerate from weeks to under 48 hours the calculation of differences in binding free energy and its application to Src homology 2 (SH2) protein cell signalling domains. The method of calculation, thermodynamic integration, is briefly outlined and we indicate how the calculation process works from the perspective of an application scientist using either the UK National Grid Service or the US Teragrid. The development of a PDA-based steering client is especially useful as it gives the application scientist more freedom. Finally, we discuss our experience in developing and deploying the application on a grid.

@inproceedings{Fowler2004,
abstract = {We describe a grid-based method to dramatically accelerate from weeks to under 48 hours the calculation of differences in binding free energy and its application to Src homology 2 (SH2) protein cell signalling domains. The method of calculation, thermodynamic integration, is briefly outlined and we indicate how the calculation process works from the perspective of an application scientist using either the UK National Grid Service or the US Teragrid. The development of a PDA-based steering client is especially useful as it gives the application scientist more freedom. Finally, we discuss our experience in developing and deploying the application on a grid.},
author = {Fowler, Philip W and Coveney, P V and Jha, S and Wan, S},
booktitle = {Proceedings of the UK e-Science All Hands Meeting},
title = {{Exact calculation of peptide-protein binding energies by steered thermodynamic integration using high performance computing grids}},
url = {http://www.allhands.org.uk/2004/proceedings/papers/154.pdf},
year = {2004}
}

### 1998

• P. V. Coveney, J. -B. Maillet, J. L. Wilson, P. W. Fowler, O. Al-Mushadani, and B. M. Boghosian, “Lattice-Gas Simulations of Ternary Amphiphilic Fluid Flow in Porous Media,” Int J Mod Phys C, vol. 9, iss. 8, pp. 1479-1490, 1998.

We develop our existing two-dimensional lattice-gas model to simulate the flow of single-phase, binary-immiscible and ternary-amphiphilic fluids. This involves the inclusion of fixed obstacles on the lattice, together with the inclusion of “no-slip” boundary conditions. Here we report on preliminary applications of this model to the flow of such fluids within model porous media. We also construct fluid invasion boundary conditions, and the effects of invading aqueous solutions of surfactant on oil-saturated rock during imbibition and drainage are described.

@article{Coveney1998,
abstract = {We develop our existing two-dimensional lattice-gas model to simulate the flow of single-phase, binary-immiscible and ternary-amphiphilic fluids. This involves the inclusion of fixed obstacles on the lattice, together with the inclusion of no-slip'' boundary conditions. Here we report on preliminary applications of this model to the flow of such fluids within model porous media. We also construct fluid invasion boundary conditions, and the effects of invading aqueous solutions of surfactant on oil-saturated rock during imbibition and drainage are described.},
author = {Coveney, P V and Maillet, J. -B. and Wilson, J L and Fowler, P W and Al-Mushadani, O. and Boghosian, B M},
doi = {10.1142/S0129183198001345},
journal = {{Int J Mod Phys C}},
number = {8},
pages = {1479-1490},
title = {{Lattice-Gas Simulations of Ternary Amphiphilic Fluid Flow in Porous Media}},
url = {http://arxiv.org/abs/comp-gas/9810002},
volume = {9},
year = {1998}
}