# Research

1. Antibiotics. I am focussing on two ways of improving the effectiveness of antibiotics.

1.1. Improving the oral bioavailability of hydrophilic drugs [1, 2, 3].

Drugs taken orally must either passively diffuse across the membranes of the epithelial cells that line the gut, in which case they must be lipophilic. Or they must  be shuttled across by an endogenous transporter, in which case they must resemble a nutrient.  Little has been known about the endogenous transporters until recently. As a consequence drugs have been optimised to be as lipophilic as possible, restricting the development of naturally-derived drugs, including antibiotics, which tend to be hydrophilic.

I am focussing on the human nutrient transporter PepT1 which transports di- and tri-peptides across the lining of the small intestine. PepT1 also happens to recognise and transport many beta-lactam antibiotics and a range of other hydrophilic drugs, thereby ensuring they have high oral bioavailabilities.

The aim of this project is to computationally predict chemical modifications for a range of antibiotics (and other drugs) that will improve their transport by hPepT1 (a peptide transporter found in the human gut) and hence improve their oral bioavailability and clinical effectiveness.

I am mainly collaborating with (Biochemistry, Oxford), and am also working with (NIH), (Biochemistry, Oxford) and (Biological and Medical Sciences, Oxford Brookes).

I am also co-supervising , a DPhil student, who working on this problem.

1.2 : predicting antibiotic susceptibility from whole genome sequencing using a crowd sourced computer network

Antibiotics are vital for the treatment of a wide range of human ailments and accounted for 6.7% of all prescriptions dispensed in the UK in the decade 2001-2011. The extensive global use of antibiotics in the last 70 years has led to bacteria developing resistance to their action. Despite this threat to the effectiveness of antibiotics, the number of new antibiotics developed has dropped markedly over the past 50 years.

Testing whether a bacterial infection is susceptible or resistant to antibiotics is based on microbiology techniques and can days to weeks, depending on the organism involved. It has been recognised that sequencing the genome of the infection-causing organism could provide a faster (and ultimately cheaper) method for clinicians to determine which antibiotic is most appropriate to treat a given infection. Although the majority of resistant phenotypes can be identified by comparing the sequence to a database of known resistance-causing mutations, rapidly predicting the phenotype of unknown mutations is an important ability.

The aim of this project is to develop a workflow that will take a novel mutation, and then, by running a range of computational simulations of the proteins involved, predict the effect of the mutation on a range of antibiotics.

To provide the computer resource required I am building a -based volunteer computing network (, like ) in collaboration with (OeRC, Oxford). I am collaborating with and at the Nuffield Department of Medicine, Oxford.

2. Cell signalling [4]

Cells are able to receive and act on external signals; how they do this is a difficult problem. The Ras proteins have long been identified as playing an important role in a wide range of cell signalling cascades. I am studying the localisation of Ras proteins to different lipid bilayers and what modulates their binding. This is a difficult non-equilibrium process and spans a large range of length and time-scales

3. Transporters [5, 6]

Transporters are membrane proteins that are able to move molecules across the cell membrane, often up their concentration gradient. There are a range of different families; I have studied how ABC transporters use ATP to drive the transport cycle and showed that energy is stored during the cycle as a structural strain which is unwound later, thereby powering the second step of the transport cycle. More recently I have studied the largest family of transporters, the Major Facilitator Superfamily (PepT1 mentioned above belongs to this family).

4. Ion Channels

Ion channels are exquisitely-sensitive protein machines which selectively conduct ions at often high rates and are able to open and close their pores in response to a wide range of stimuli. Molecular simulation is a vital way of improving our understanding of these subtle proteins. I have studied how  potassium ion channels permit potassium to pass, but not sodium, [7], how they can conduct ions at such fast rates [8, 9] and how voltage-gated potassium channels open and close in response to changes in the transmembrane potential [10]. Finally I have had a long-standing collaboration on an inwardly-rectifying potassium channel, Kir1.1, with  and  [11, 12, 13, 14, 15]

5. e-Infrastructure and grid computing

I have had a long interest in developing novel methods for accelerating calculations using grid computing [16, 17, 18, 19]. I am also a Co-Investigator on the  whose aim is to encourage the use of e-Infrastructure within the UK academic community.

[1] 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.
@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}
}
[2] 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.
@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}
}
[3] 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.
@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.}
}
[4] 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.
@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}
}
[5] 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.
@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}
}
[6] 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.
@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}
}
[7] 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.
@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}
}
[8] 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.
@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}
}
[9] 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.
@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}
}
[10] 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.
@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}
}
[11] 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.
@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}
}
[12] 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.
@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}
}
[13] 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.
@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}
}
[14] 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.
@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}
}
[15] 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.
@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}
}
[16] 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.
@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}
}
[17] 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.
@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}
}
[18] 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.
@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}
}
[19] 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.
@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}
}