Dr Philip Fowler

Research Area: Bioinformatics & Stats (inc. Modelling and Computational Biology)
Technology Exchange: Bioinformatics, Computational biology, Drug discovery and Protein interaction
Scientific Themes: Immunology & Infectious Disease and Protein Science & Structural Biology
Keywords: antibiotic resistance, tuberculosis, clinical microbiology, computational biophysics, molecular dynamics, protein structure, protein-drug interactions, free energy methods, distributed computing, volunteer computing and open science
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My research aims to develop computational methods able to predict antibiotic resistance by considering the structures of the target protein, the antibiotic and how they dynamically interact  with one another.

The Modernising Medical Microbiology group here in Oxford is pioneering genetics-based clinical microbiology. The central idea is to infer which antibiotics can be used to treat an infection by examining the mutations in the genome and looking up their effect in a catalogue of previously-seen cases. Here predictive methods are essential if we are to deal with cases that have novel or rare mutations. 

Alternatively, predictive methods could be used in the development of new antibiotics (or the modification of existing ones) to determine how many mutations allow the bacteria to escape the action of the drug. Minimising this number should, we hope, prolong the lifespan of an antibiotic.

My hypothesis is that mutations in an open-reading frame that cause resistance do so by reducing how well the antibiotic binds to its target protein, whilst not altering how well the natural substrate binds. Prediction is therefore a matter of determining how the binding free energies of both molecules change upon introduction of the protein mutation.

Calculating small molecule-protein binding free energies is a mature field. Probably the best-known approach is computational docking which uses a simple heuristic functional to estimate how well a small molecule binds in a specific orientation to a protein. Whilst fast, these methods take no account of the dynamics of either protein or drug are not accurate for a problem of this subtlety. 

Instead I am applying a class of methods derived from statistical mechanics over sixty years ago. They require, however, 4-5 orders of magnitude more computational resource than simple computational docking and, as a result, have only recently begun to find application outside of theoretical physical chemistry.

My research will benefit from the large amount of genomic and drug susceptibility data being collected by the CRyPTIC project, which is lead by the University of Oxford. I also have interests in distributed computing and citizen science and will be launching a project, called bashthebug.net, in 2017.

I lecture the first-year biochemistry undergraduates on protein folding, which I was awarded an Excellent Teacher award in 2013. I'm also a qualified Software Carpentry Instructor and have run several bootcamps, both here in Oxford and elsewhere. Starting in October 2016, I am also lecturing on the Programming Course for the Doctoral Training Centre and coordinating the Bioinformatics Course. Finally I am Chair of the Advanced Research Computing (ARC) Advisory Board which provides strategic direction for the central University high performance computing facility.

Name Department Institution Country
Professor Derrick Crook Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Professor (Ann) Sarah Walker Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Professor Tim Peto Experimental Medicine Division Oxford University, John Radcliffe Hospital United Kingdom
Dr David Wallom (MPLS) Oxford University, Oxford e-Research Centre United Kingdom
Associate Professor Simon Newstead Biochemistry University of Oxford United Kingdom
Fowler PW, Sansom MS, Reithmeier RA. 2017. Effect of the Southeast Asian Ovalocytosis Deletion on the Conformational Dynamics of Signal-Anchor Transmembrane Segment 1 of Red Cell Anion Exchanger 1 (AE1, Band 3, or SLC4A1). Biochemistry, 56 (5), pp. 712-722. | Show Abstract | Read more

The first transmembrane (TM1) helix in the red cell anion exchanger (AE1, Band 3, or SLC4A1) acts as an internal signal anchor that binds the signal recognition particle and directs the nascent polypeptide chain to the endoplasmic reticulum (ER) membrane where it moves from the translocon laterally into the lipid bilayer. The sequence N-terminal to TM1 forms an amphipathic helix that lies at the membrane interface and is connected to TM1 by a bend at Pro403. Southeast Asian ovalocytosis (SAO) is a red cell abnormality caused by a nine-amino acid deletion (Ala400-Ala408) at the N-terminus of TM1. Here we demonstrate, by extensive (∼4.5 μs) molecular dynamics simulations of TM1 in a model 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane, that the isolated TM1 peptide is highly dynamic and samples the structure of TM1 seen in the crystal structure of the membrane domain of AE1. The SAO deletion not only removes the proline-induced bend but also causes a "pulling in" of the part of the amphipathic helix into the hydrophobic phase of the bilayer, as well as the C-terminal of the peptide. The dynamics of the SAO peptide very infrequently resembles the structure of TM1 in AE1, demonstrating the disruptive effect the SAO deletion has on AE1 folding. These results provide a precise molecular view of the disposition and dynamics of wild-type and SAO TM1 in a lipid bilayer, an important early biosynthetic intermediate in the insertion of AE1 into the ER membrane, and extend earlier results of cell-free translation experiments.

Koldsø H, Reddy T, Fowler PW, Duncan AL, Sansom MS. 2016. Membrane Compartmentalization Reducing the Mobility of Lipids and Proteins within a Model Plasma Membrane. J Phys Chem B, 120 (34), pp. 8873-8881. | Show Abstract | Read more

The cytoskeleton underlying cell membranes may influence the dynamic organization of proteins and lipids within the bilayer by immobilizing certain transmembrane (TM) proteins and forming corrals within the membrane. Here, we present coarse-grained resolution simulations of a biologically realistic membrane model of asymmetrically organized lipids and TM proteins. We determine the effects of a model of cytoskeletal immobilization of selected membrane proteins using long time scale coarse-grained molecular dynamics simulations. By introducing compartments with varying degrees of restraints within the membrane models, we are able to reveal how compartmentalization caused by cytoskeletal immobilization leads to reduced and anomalous diffusional mobility of both proteins and lipids. This in turn results in a reduced rate of protein dimerization within the membrane and of hopping of membrane proteins between compartments. These simulations provide a molecular realization of hierarchical models often invoked to explain single-molecule imaging studies of membrane proteins.

Fowler PW, Williamson JJ, Sansom MS, Olmsted PD. 2016. Roles of Interleaflet Coupling and Hydrophobic Mismatch in Lipid Membrane Phase-Separation Kinetics. J Am Chem Soc, 138 (36), pp. 11633-11642. | Show Abstract | Read more

Characterizing the nanoscale dynamic organization within lipid bilayer membranes is central to our understanding of cell membranes at a molecular level. We investigate phase separation and communication across leaflets in ternary lipid bilayers, including saturated lipids with between 12 and 20 carbons per tail. Coarse-grained molecular dynamics simulations reveal a novel two-step kinetics due to hydrophobic mismatch, in which the initial response of the apposed leaflets upon quenching is to increase local asymmetry (antiregistration), followed by dominance of symmetry (registration) as the bilayer equilibrates. Antiregistration can become thermodynamically preferred if domain size is restricted below ∼20 nm, with implications for the symmetry of rafts and nanoclusters in cell membranes, which have similar reported sizes. We relate our findings to theory derived from a semimicroscopic model in which the leaflets experience a "direct" area-dependent coupling, and an "indirect" coupling that arises from hydrophobic mismatch and is most important at domain boundaries. Registered phases differ in composition from antiregistered phases, consistent with a direct coupling between the leaflets. Increased hydrophobic mismatch purifies the phases, suggesting that it contributes to the molecule-level lipid immiscibility. Our results demonstrate an interplay of competing interleaflet couplings that affect phase compositions and kinetics, and lead to a length scale that can influence lateral and transverse bilayer organization within cells.

Fowler PW, Hélie J, Duncan A, Chavent M, Koldsø H, Sansom MS. 2016. Membrane stiffness is modified by integral membrane proteins. Soft Matter, 12 (37), pp. 7792-7803. | Show Abstract | Read more

The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, Kc). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.

Samsudin F, Parker JL, Sansom MSP, Newstead S, Fowler PW. 2016. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter. Cell Chem Biol, 23 (2), pp. 299-309. | Show Abstract | Read more

Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the ?-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepT(St), and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theoretically exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homology model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families.

Beale JH, Parker JL, Samsudin F, Barrett AL, Senan A, Bird LE, Scott D, Owens RJ, Sansom MS, Tucker SJ et al. 2015. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport. Structure, 23 (10), pp. 1889-1899. | Show Abstract | Read more

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Jefferys E, Sands ZA, Shi J, Sansom MS, Fowler PW. 2015. Alchembed: A Computational Method for Incorporating Multiple Proteins into Complex Lipid Geometries. J Chem Theory Comput, 11 (6), pp. 2743-2754. | Show Abstract | Read more

A necessary step prior to starting any membrane protein computer simulation is the creation of a well-packed configuration of protein(s) and lipids. Here, we demonstrate a method, alchembed, that can simultaneously and rapidly embed multiple proteins into arrangements of lipids described using either atomistic or coarse-grained force fields. During a short simulation, the interactions between the protein(s) and lipids are gradually switched on using a soft-core van der Waals potential. We validate the method on a range of membrane proteins and determine the optimal soft-core parameters required to insert membrane proteins. Since all of the major biomolecular codes include soft-core van der Waals potentials, no additional code is required to apply this method. A tutorial is included in the Supporting Information.

Beale JH, Parker JL, Samsudin F, Barrett AL, Senan A, Bird LE, Scott D, Owens RJ, Sansom MSP, Tucker SJ et al. 2015. Crystal structures of the extracellular domain from PepT1 and PepT2 provide novel insights into mammalian peptide transport Structure, 23 (10), pp. 1889-1899. | Show Abstract | Read more

© 2015 The Authors. Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Reddy T, Shorthouse D, Parton DL, Jefferys E, Fowler PW, Chavent M, Baaden M, Sansom MS. 2015. Nothing to sneeze at: a dynamic and integrative computational model of an influenza A virion. Structure, 23 (3), pp. 584-597. | Show Abstract | Read more

The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data from X-ray crystallography, nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and lipidomics to build a model of the intact influenza A virion. This is the basis of microsecond-scale coarse-grained molecular dynamics simulations of the virion, providing simulations at different temperatures and with varying lipid compositions. The presence of the Forssman glycolipid alters a number of biophysical properties of the virion, resulting in reduced mobility of bilayer lipid and protein species. Reduced mobility in the virion membrane may confer physical robustness to changes in environmental conditions. Our simulations indicate that viral spike proteins do not aggregate and thus are competent for multivalent immunoglobulin G interactions.

Fowler PW, Orwick-Rydmark M, Radestock S, Solcan N, Dijkman PM, Lyons JA, Kwok J, Caffrey M, Watts A, Forrest LR, Newstead S. 2015. Gating topology of the proton-coupled oligopeptide symporters. Structure, 23 (2), pp. 290-301. | Show Abstract | Read more

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.

Reddy T, Shorthouse D, Parton D, Jefferys E, Fowler PW, Chavent M, Baaden M, Sansom MSP. 2015. Nothing to Sneeze at: A Full-Scale Computational Model of the Human Influenza Virion BIOPHYSICAL JOURNAL, 108 (2), pp. 31A-31A.

Samsudin MF, Solcan N, Newstead S, Sansom MSP, Fowler PW. 2015. Which Free Energy Methods can Predict Transport by Proton-Dependent Oligopeptide Symporters? BIOPHYSICAL JOURNAL, 108 (2), pp. 310A-310A.

Fowler PW, Koldso H, Duncan A, Sansom MSP. 2015. Investigating the Role of Bilayer Size and Composition on Membrane Fluctuations using Large Coarse-Grained Simulations BIOPHYSICAL JOURNAL, 108 (2), pp. 240A-240A.

Fowler PW, Bollepalli MK, Rapedius M, Nematian-Ardestani E, Shang L, Sansom MS, Tucker SJ, Baukrowitz T. 2014. Insights into the structural nature of the transition state in the Kir channel gating pathway. Channels (Austin), 8 (6), pp. 551-555. | Show Abstract | Read more

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 center 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.

Bollepalli MK, Fowler PW, Rapedius M, Shang L, Sansom MSP, Tucker SJ, Baukrowitz T. 2014. State-dependent network connectivity determines gating in a K+ channel Structure, 22 (7), pp. 1037-1046. | Show Abstract | Read more

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. © 2014 The Authors.

Cited:

20

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Stelzl LS, Fowler PW, Sansom MSP, Beckstein O. 2014. Flexible gates generate occluded intermediates in the transport cycle of LacY Journal of Molecular Biology, 426 (3), pp. 735-751. | Show Abstract | Read more

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, PepT So , 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. © 2013 The Authors.

Jefferys E, Sansom MS, Fowler PW. 2014. NRas slows the rate at which a model lipid bilayer phase separates. Faraday Discuss, 169 pp. 209-223. | Show Abstract | Read more

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.

Bollepalli MK, Fowler PW, Rapedius M, Shang L, Sansom MS, Tucker SJ, Baukrowitz T. 2014. State-dependent network connectivity determines gating in a K+ channel. Structure, 22 (7), pp. 1037-1046. | Show Abstract | Read more

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.

Fowler PW, Abad E, Beckstein O, Sansom MS. 2013. Energetics of Multi-Ion Conduction Pathways in Potassium Ion Channels. J Chem Theory Comput, 9 (11), pp. 5176-5189. | Show Abstract | Read more

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.

Fowler PW, Beckstein O, Abad E, Sansom MS. 2013. Detailed Examination of a Single Conduction Event in a Potassium Channel. J Phys Chem Lett, 4 (18), pp. 3104-3109. | Show Abstract | Read more

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.

Stelzl LS, Fowler PW, Sansom MS, Beckstein O. 2014. Flexible gates generate occluded intermediates in the transport cycle of LacY. J Mol Biol, 426 (3), pp. 735-751. | Show Abstract | Read more

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.

Fowler PW, Sansom MS. 2013. The pore of voltage-gated potassium ion channels is strained when closed. Nat Commun, 4 pp. 1872. | Show Abstract | Read more

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) α-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.

Solcan N, Kwok J, Fowler PW, Cameron AD, Drew D, Iwata S, Newstead S. 2012. Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J, 31 (16), pp. 3411-3421. | Show Abstract | Read more

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-Å 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.

Cited:

97

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Solcan N, Kwok J, Fowler PW, Cameron AD, Drew D, Iwata S, Newstead S. 2012. Alternating access mechanism in the POT family of oligopeptide transporters EMBO Journal, 31 (16), pp. 3411-3421. | Show Abstract | Read more

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-Å 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. © 2012 European Molecular Biology Organization.

Cited:

143

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Newstead S, Drew D, Cameron AD, Postis VLG, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MSP, McPherson MJ et al. 2011. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2 EMBO Journal, 30 (2), pp. 417-426. | Show Abstract | Read more

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 Å 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. © 2011 European Molecular Biology Organization | All Rights Reserved.

Newstead S, Drew D, Cameron AD, Postis VL, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MS, McPherson MJ et al. 2011. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J, 30 (2), pp. 417-426. | Show Abstract | Read more

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 Å 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.

Paynter JJ, Andres-Enguix I, Fowler PW, Tottey S, Cheng W, Enkvetchakul D, Bavro VN, Kusakabe Y, Sansom MS, Robinson NJ et al. 2010. Functional complementation and genetic deletion studies of KirBac channels: activatory mutations highlight gating-sensitive domains. J Biol Chem, 285 (52), pp. 40754-40761. | Show Abstract | Read more

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is 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 Escherichia coli and Saccharomyces 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 favor 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.

Paynter JJ, Andres-Enguix I, Fowler PW, Tottey S, Cheng W, Enkvetchakul D, Bavro VN, Kusakabe Y, Sansom MSP, Robinson NJ et al. 2010. Functional Complementation and Genetic Deletion Studies of KirBac Channels Journal of Biological Chemistry, 285 (52), pp. 40754-40761. | Show Abstract | Read more

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is 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 Escherichia coli and Saccharomyces 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 favor 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Newstead S, Fowler PW, Bilton P, Carpenter EP, Sadler PJ, Campopiano DJ, Sansom MS, Iwata S. 2009. Insights into how nucleotide-binding domains power ABC transport. Structure, 17 (9), pp. 1213-1222. | Show Abstract | Read more

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.

Fowler PW, Tai K, Sansom MS. 2008. The selectivity of K+ ion channels: testing the hypotheses. Biophys J, 95 (11), pp. 5062-5072. | Show Abstract | Read more

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.

Psachoulia E, Fowler PW, Bond PJ, Sansom MS. 2008. Helix-helix interactions in membrane proteins: coarse-grained simulations of glycophorin a helix dimerization. Biochemistry, 47 (40), pp. 10503-10512. | Show Abstract | Read more

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.

Rapedius M, Paynter JJ, Fowler PW, Shang L, Sansom MS, Tucker SJ, Baukrowitz T. 2007. Control of pH and PIP2 gating in heteromeric Kir4.1/Kir5.1 channels by H-Bonding at the helix-bundle crossing. Channels (Austin), 1 (5), pp. 327-330. | Show Abstract | Read more

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.

Rapedius M, Fowler PW, Shang L, Sansom MS, Tucker SJ, Baukrowitz T. 2007. H bonding at the helix-bundle crossing controls gating in Kir potassium channels. Neuron, 55 (4), pp. 602-614. | Show Abstract | Read more

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.

Fowler PW, Balali-Mood K, Deol S, Coveney PV, Sansom MS. 2007. Monotopic enzymes and lipid bilayers: a comparative study. Biochemistry, 46 (11), pp. 3108-3115. | Show Abstract | Read more

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.

Fowler PW, Williamson JJ, Sansom MS, Olmsted PD. 2016. Roles of Interleaflet Coupling and Hydrophobic Mismatch in Lipid Membrane Phase-Separation Kinetics. J Am Chem Soc, 138 (36), pp. 11633-11642. | Show Abstract | Read more

Characterizing the nanoscale dynamic organization within lipid bilayer membranes is central to our understanding of cell membranes at a molecular level. We investigate phase separation and communication across leaflets in ternary lipid bilayers, including saturated lipids with between 12 and 20 carbons per tail. Coarse-grained molecular dynamics simulations reveal a novel two-step kinetics due to hydrophobic mismatch, in which the initial response of the apposed leaflets upon quenching is to increase local asymmetry (antiregistration), followed by dominance of symmetry (registration) as the bilayer equilibrates. Antiregistration can become thermodynamically preferred if domain size is restricted below ∼20 nm, with implications for the symmetry of rafts and nanoclusters in cell membranes, which have similar reported sizes. We relate our findings to theory derived from a semimicroscopic model in which the leaflets experience a "direct" area-dependent coupling, and an "indirect" coupling that arises from hydrophobic mismatch and is most important at domain boundaries. Registered phases differ in composition from antiregistered phases, consistent with a direct coupling between the leaflets. Increased hydrophobic mismatch purifies the phases, suggesting that it contributes to the molecule-level lipid immiscibility. Our results demonstrate an interplay of competing interleaflet couplings that affect phase compositions and kinetics, and lead to a length scale that can influence lateral and transverse bilayer organization within cells.

Fowler PW, Hélie J, Duncan A, Chavent M, Koldsø H, Sansom MS. 2016. Membrane stiffness is modified by integral membrane proteins. Soft Matter, 12 (37), pp. 7792-7803. | Show Abstract | Read more

The ease with which a cell membrane can bend and deform is important for a wide range of biological functions. Peripheral proteins that induce curvature in membranes (e.g. BAR domains) have been studied for a number of years. Little is known, however, about the effect of integral membrane proteins on the stiffness of a membrane (characterised by the bending rigidity, Kc). We demonstrate by computer simulation that adding integral membrane proteins at physiological densities alters the stiffness of the membrane. First we establish that the coarse-grained MARTINI forcefield is able to accurately reproduce the bending rigidity of a small patch of 1500 phosphatidyl choline lipids by comparing the calculated value to both experiment and an atomistic simulation of the same system. This enables us to simulate the dynamics of large (ca. 50 000 lipids) patches of membrane using the MARTINI coarse-grained description. We find that altering the lipid composition changes the bending rigidity. Adding integral membrane proteins to lipid bilayers also changes the bending rigidity, whilst adding a simple peripheral membrane protein has no effect. Our results suggest that integral membrane proteins can have different effects, and in the case of the bacterial outer membrane protein, BtuB, the greater the density of protein, the larger the reduction in stiffness.

Samsudin F, Parker JL, Sansom MSP, Newstead S, Fowler PW. 2016. Accurate Prediction of Ligand Affinities for a Proton-Dependent Oligopeptide Transporter. Cell Chem Biol, 23 (2), pp. 299-309. | Show Abstract | Read more

Membrane transporters are critical modulators of drug pharmacokinetics, efficacy, and safety. One example is the proton-dependent oligopeptide transporter PepT1, also known as SLC15A1, which is responsible for the uptake of the ?-lactam antibiotics and various peptide-based prodrugs. In this study, we modeled the binding of various peptides to a bacterial homolog, PepT(St), and evaluated a range of computational methods for predicting the free energy of binding. Our results show that a hybrid approach (endpoint methods to classify peptides into good and poor binders and a theoretically exact method for refinement) is able to accurately predict affinities, which we validated using proteoliposome transport assays. Applying the method to a homology model of PepT1 suggests that the approach requires a high-quality structure to be accurate. Our study provides a blueprint for extending these computational methodologies to other pharmaceutically important transporter families.

Beale JH, Parker JL, Samsudin F, Barrett AL, Senan A, Bird LE, Scott D, Owens RJ, Sansom MS, Tucker SJ et al. 2015. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport. Structure, 23 (10), pp. 1889-1899. | Show Abstract | Read more

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Reddy T, Shorthouse D, Parton DL, Jefferys E, Fowler PW, Chavent M, Baaden M, Sansom MS. 2015. Nothing to sneeze at: a dynamic and integrative computational model of an influenza A virion. Structure, 23 (3), pp. 584-597. | Show Abstract | Read more

The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data from X-ray crystallography, nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and lipidomics to build a model of the intact influenza A virion. This is the basis of microsecond-scale coarse-grained molecular dynamics simulations of the virion, providing simulations at different temperatures and with varying lipid compositions. The presence of the Forssman glycolipid alters a number of biophysical properties of the virion, resulting in reduced mobility of bilayer lipid and protein species. Reduced mobility in the virion membrane may confer physical robustness to changes in environmental conditions. Our simulations indicate that viral spike proteins do not aggregate and thus are competent for multivalent immunoglobulin G interactions.

Fowler PW, Abad E, Beckstein O, Sansom MS. 2013. Energetics of Multi-Ion Conduction Pathways in Potassium Ion Channels. J Chem Theory Comput, 9 (11), pp. 5176-5189. | Show Abstract | Read more

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.

Fowler PW, Sansom MS. 2013. The pore of voltage-gated potassium ion channels is strained when closed. Nat Commun, 4 pp. 1872. | Show Abstract | Read more

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) α-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.

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Newstead S, Drew D, Cameron AD, Postis VLG, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MSP, McPherson MJ et al. 2011. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2 EMBO Journal, 30 (2), pp. 417-426. | Show Abstract | Read more

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 Å 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. © 2011 European Molecular Biology Organization | All Rights Reserved.

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