Megan O'Mara

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae

Publisher: Wiley

Date: 16-01-2014

DOI: 10.1111/MMI.12504

Abstract: Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Encyclopedia of Computational Neuroscience

Publisher: Springer New York

Date: 2013

DOI: 10.1007/978-1-4614-7320-6

Probing the Pharmacological Binding Sites of P-Glycoprotein Using Umbrella Sampling Simulations

Publisher: American Chemical Society (ACS)

Date: 12-12-2019

DOI: 10.1021/ACS.JCIM.8B00624

Abstract: The human multidrug transporter P-glycoprotein (P-gp) transports over 200 chemically erse substrates, influencing their bioavailability and tissue distribution. Pharmacological studies have identified both competitive and noncompetitive P-gp substrates, but neither the precise location of the substrate binding sites, nor the basis of competitive and noncompetitive interactions has been fully characterized. Here, potential of mean force (PMF) calculations are used to identify the transport-competent minimum free energy binding locations of five compounds, Hoechst 33342, Rhodamine 123, paclitaxel, tariquidar, and verapamil to P-gp. Unrestrained molecular dynamics simulations were also performed to confirm the substrates were stable in the energy wells determined using the PMF calculations. All compounds had energy minima within the P-gp transmembrane (TM) pore. For Hoechst 33342 and Rhodamine 123, a second minimum outside the TM pore was also identified. Based on this and previous studies of nicardipine and morphine [ Subramanian et al. J. Chem. Inf. Model. 2015 , 55 , 1202 ], a general scheme that accounts for the observed noncompetitive and competitive substrate interactions with P-gp is proposed.

Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.BPC.2015.08.004

Abstract: Prokaryotic metal-ion receptor proteins, or solute-binding proteins, facilitate the acquisition of metal ions from the extracellular environment. Pneumococcal surface antigen A (PsaA) is the primary Mn(2+)-recruiting protein of the human pathogen Streptococcus pneumoniae and is essential for its in vivo colonization and virulence. The recently reported high-resolution structures of metal-free and metal-bound PsaA have provided the first insights into the mechanism of PsaA-facilitated metal binding. However, the conformational dynamics of metal-free PsaA in solution remain unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also s les structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is both larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomic-resolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution.

ABC transporters: a riddle wrapped in a mystery inside an enigma

Publisher: Elsevier BV

Date: 10-2009

DOI: 10.1016/J.TIBS.2009.06.004

Abstract: ATP-binding cassette (ABC) transporters form one of the largest and most ancient of protein families. ABC transporters couple hydrolysis of ATP to vectorial translocation of erse substrates across cellular membranes. Many human ABC transporters are medically important in causing, for ex le, multidrug resistance to cytotoxic drugs. Seven complete prokaryotic structures and one eukaryotic structure have been solved for transporters from 2002 to date, and a wealth of research is being conducted on and around these structures to resolve the mechanistic conundrum of how these transporters couple ATP hydrolysis in cytosolic domains to substrate translocation through the transmembrane pore. Many questions remained unanswered about this mechanism, despite a plethora of data and a number of interesting and controversial models.

Identification of Possible Binding Sites for Morphine and Nicardipine on the Multidrug Transporter P-Glycoprotein Using Umbrella Sampling Techniques

Publisher: American Chemical Society (ACS)

Date: 18-05-2015

DOI: 10.1021/CI5007382

Abstract: The multidrug transporter P-glycoprotein (P-gp) is central to the development of multidrug resistance in cancer. While residues essential for transport and binding have been identified, the location, composition, and specificity of potential drug binding sites are uncertain. Here molecular dynamics simulations are used to calculate the free energy profile for the binding of morphine and nicardipine to P-gp. We show that morphine and nicardipine primarily interact with key residues implicated in binding and transport from mutational studies, binding at different but overlapping sites within the transmembrane pore. Their permeation pathways were distinct but involved overlapping sets of residues. The results indicate that the binding location and permeation pathways of morphine and nicardipine are not well separated and cannot be considered as unique. This has important implications for our understanding of substrate uptake and transport by P-gp. Our results are independent of the choice of starting structure and consistent with a range of experimental studies.

The net electrostatic potential and hydration of ABCG2 affect substrate transport

Publisher: Springer Science and Business Media LLC

Date: 18-08-2023

DOI: 10.1038/S41467-023-40610-5

Abstract: ABCG2 is a medically important ATP-binding cassette transporter with crucial roles in the absorption and distribution of chemically- erse toxins and drugs, reducing the cellular accumulation of chemotherapeutic drugs to facilitate multidrug resistance in cancer. ABCG2’s capacity to transport both hydrophilic and hydrophobic compounds is not well understood. Here we assess the molecular basis for substrate discrimination by the binding pocket. Substitution of a phylogenetically-conserved polar residue, N436, to alanine in the binding pocket of human ABCG2 permits only hydrophobic substrate transport, revealing the unique role of N436 as a discriminator. Molecular dynamics simulations show that this alanine substitution alters the electrostatic potential of the binding pocket favoring hydration of the transport pore. This change affects the contact with substrates and inhibitors, abrogating hydrophilic compound transport while retaining the transport of hydrophobic compounds. The N436 residue is also required for optimal transport inhibition of ABCG2, as many inhibitors are functionally impaired by this ABCG2 mutation. Overall, these findings have biomedical implications, broadly extending our understanding of substrate and inhibitor interactions.

Dynamics of the Acinetobacter baumannii inner membrane under exogenous polyunsaturated fatty acid stress

Publisher: Elsevier BV

Date: 07-2022

DOI: 10.1016/J.BBAMEM.2022.183908

Abstract: Exogenous polyunsaturated fatty acids (PUFAs) are readily incorporated into the synthesis pathways of A. baumannii membrane phospholipids, where they contribute to reduced bacterial fitness and increased antimicrobial susceptibility. Here we examine the impact of PUFA membrane modification on membrane organisation and biophysical properties using coarse grained MARTINI simulations of chemically representative membrane models developed from mass-spectrometry datasets of an untreated, arachidonic acid (AA) treated and docosahexaenoic acid (DHA) treated A. baumannii membranes. Enzymatic integration of AA or DHA into phospholipids of the A. baumannii membrane resulted in modulation of membrane biophysical properties. Membrane thickness decreased slightly following PUFA treatment, concomitant with changes in the lateral area per lipid of each lipid headgroup class. PUFA treatment resulted in a decrease in membrane ordering and an increase in lipid lateral diffusion. Changes in lateral membrane organisation were observed in the PUFA treated membranes, with a concurrent increase in ordered cardiolipin domains and disordered PUFA-containing domains. Notably, separation between ordered and disordered domains was enhanced and was more pronounced for DHA relative to AA, providing a possible mechanism for greater antimicrobial action of DHA relative to AA observed experimentally. Furthermore, the membrane active antimicrobial, pentamidine, preferentially adsorbs to cardiolipin domains of the A. baumannii model membranes. This interaction, and membrane penetration of pentamidine, was enhanced following PUFA treatment. Cumulatively, this work explores the wide-ranging effects of PUFA incorporation on the A. baumannii membrane and provides a molecular basis for bacterial inner membrane disruption by PUFAs.

Structural Alterations in a Rat Model of Short-Term Conductive Hearing Loss Are Associated With Reduced Resting State Functional Connectivity

Publisher: Frontiers Media SA

Date: 12-08-2021

DOI: 10.3389/FNSYS.2021.655172

Abstract: Conductive hearing loss (CHL) results in attenuation of air conducted sound reaching the inner ear. How a change in air conducted sound alters the auditory system resulting in cortical alterations is not well understood. Here, we have assessed structural and functional magnetic resonance imaging (MRI) in an adult (P60) rat model of short-term conductive hearing loss (1 week). Diffusion tensor imaging (DTI) revealed fractional anisotropy (FA) and axial diffusivity alterations after hearing loss that circumscribed the auditory cortex (AC). Tractography found the lateral lemniscus tract leading to the bilateral inferior colliculus (IC) was reduced. For baseline comparison, DTI and tractography alterations were not found for the somatosensory cortex. To determine functional connectivity changes due to hearing loss, seed-based analysis (SBA) and independent component analysis (ICA) were performed. Short term conductive hearing loss altered functional connectivity in the AC and IC, but not the somatosensory cortex. The results present an exploratory neuroimaging assessment of structural alterations coupled to a change in functional connectivity after conductive hearing loss. The results and implications for humans consist of structural-functional brain alterations following short term hearing loss in adults.

Understanding the accumulation of P-glycoprotein substrates within cells: The effect of cholesterol on membrane partitioning

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.BBAMEM.2015.12.025

Abstract: The apparent activity of the multidrug transporter P-glycoprotein (P-gp) is enhanced by the presence of cholesterol. Whether this is due to the direct effect of cholesterol on the activity of P-gp, its effect on the local concentration of substrate in the membrane, or its effect on the rate of entry of the drug into the cell, is unknown. In this study, molecular dynamics simulation techniques coupled with potential of mean force calculations have been used to investigate the role of cholesterol in the movement of four P-gp substrates across a POPC bilayer in the presence or absence of 10% cholesterol. The simulations suggest that the presence of cholesterol lowers the free energy associated with entering the middle of the bilayer in a substrate-specific manner. These findings suggest that P-gp substrates may preferentially accumulate in cholesterol-rich regions of the membrane, which may explain its enhanced transport activity.

The Fats of Life: Using Computational Chemistry to Characterise the Eukaryotic Cell Membrane

Publisher: CSIRO Publishing

Date: 2020

DOI: 10.1071/CH19353

Abstract: Our current knowledge of the structural dynamics and complexity of lipid bilayers is still developing. Computational techniques, especially molecular dynamics simulations, have increased our understanding significantly as they allow us to model functions that cannot currently be experimentally resolved. Here we review available computational tools and techniques, the role of the major lipid species, insights gained into lipid bilayer structure and function from molecular dynamics simulations, and recent progress towards the computational modelling of the physiological complexity of eukaryotic lipid bilayers.

Structural and dynamic perspectives on the promiscuous transport activity of P-glycoprotein

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.NEUINT.2016.05.005

Abstract: The multidrug transporter P-glycoprotein (P-gp) is expressed in the blood-brain barrier endothelium where it effluxes a range of drug substrates, preventing their accumulation within the brain. P-gp has been studied extensively for 40 years because of its crucial role in the absorption, distribution, metabolism and elimination of a range of pharmaceutical compounds. Despite this, many aspects of the structure-function mechanism of P-gp are unresolved. Here we review the emerging role of molecular dynamics simulation techniques in our understanding of the membrane-embedded conformation of P-gp. We discuss its conformational plasticity in the presence and absence of ATP, and recent efforts to characterize the drug binding sites and uptake pathways.

Synthetically controlling dendrimer flexibility improves delivery of large plasmid DNA

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C7SC00097A

Abstract: Tools for editing the genome and epigenome have revolutionised the field of molecular biology and represent a new frontier in targeted therapeutic intervention.

Functional magnetic resonance imaging of enhanced central auditory gain and electrophysiological correlates in a behavioral model of hyperacusis

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1016/J.HEARES.2020.107908

Hydroxy Groups Enhance [2]Rotaxane Anion Binding Selectivity

Publisher: Wiley

Date: 05-04-2022

DOI: 10.1002/CHEM.202200389

Abstract: We report the synthesis of two [2]rotaxanes containing an interlocked three dimensional binding cavity formed from a pyridinium bis(amide) axle component containing two phenol donors, and an isophthalamide based macrocycle. In the competitive solvent mixture 1 : 1 CDCl 3 : CD 3 OD, one of the receptors exhibits a much higher selectivity preference for chloride than an analogous rotaxane without the hydroxy groups. X‐ray crystal structures reveal the chloride anion guest encapsulated within the interlocked binding cavity, though not all of the hydrogen bond donors are utilised. Computational semi‐empirical simulations indicate that secondary intermolecular interactions occur between the axle hydroxy hydrogen bond donors and the [2]rotaxane macrocycle components, contributing to a more preorganised binding pocket, which may be responsible for the observed enhanced selectivity.

Enzyme inspired polymer functionalized with an artificial catalytic triad

Publisher: Elsevier BV

Date: 05-2021

DOI: 10.1016/J.POLYMER.2021.123735

The allosteric inhibition of glycine transporter 2 by bioactive lipid analgesics is controlled by penetration into a deep lipid cavity

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.JBC.2021.100282

Imperfect coordination chemistry facilitates metal ion release in the Psa permease

Publisher: Springer Science and Business Media LLC

Date: 10-11-2014

DOI: 10.1038/NCHEMBIO.1382

Abstract: The relative stability of alent first-row transition metal ion complexes, as defined by the Irving-Williams series, poses a fundamental chemical challenge for selectivity in bacterial metal ion acquisition. Here we show that although the substrate-binding protein of Streptococcus pneumoniae, PsaA, is finely attuned to bind its physiological substrate manganese, it can also bind a broad range of other alent transition metal cations. By combining high-resolution structural data, metal-binding assays and mutational analyses, we show that the inability of open-state PsaA to satisfy the preferred coordination chemistry of manganese enables the protein to undergo the conformational changes required for cargo release to the Psa permease. This is specific for manganese ions, whereas zinc ions remain bound to PsaA. Collectively, these findings suggest a new ligand binding and release mechanism for PsaA and related substrate-binding proteins that facilitate specificity for alent cations during competition from zinc ions, which are more abundant in biological systems.

Conserved FimH mutations in the global Escherichia coli ST131 multi-drug resistant lineage weaken interdomain interactions and alter adhesin function

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.CSBJ.2022.08.040

Polymer–solvent interactions as a tool to engineer material properties

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D1ME00111F

Abstract: Materials with multiple reversible cross-linkers will reassemble during exposure to solvent vapours altering mechanical properties even after drying.

Mechanism of activation of protein kinase JAK2 by the growth hormone receptor

Publisher: American Association for the Advancement of Science (AAAS)

Date: 16-05-2014

DOI: 10.1126/SCIENCE.1249783

Abstract: Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors.

Lipid-Based Inhibitors Act Directly on GlyT2.

Publisher: American Chemical Society (ACS)

Date: 05-12-2019

DOI: 10.1021/ACSCHEMNEURO.8B00586

Abstract: The endogenous lipids N-arachidonylglycine and oleoyl-l-carnitine are potential therapeutic leads in the treatment of chronic pain through their inhibition of the glycine transporter GlyT2. However, their mechanism of action is unknown. It has been hypothesized that these "bioactive" lipids either inhibit GlyT2 indirectly, by significantly perturbing the biophysical properties of the membrane, or directly, by binding directly to the transporter (either from a membrane-exposed or solvent-exposed binding site). Here, we used molecular dynamics simulations to study the effects of the lipids anandamide, N-arachidonylglycine, and oleoyl-l-carnitine on (a) the biophysical properties of the bilayer and (b) direct binding interactions with GlyT2. During the simulations, the biophysical properties of the bilayer itself, for ex le, the area per lipid, bilayer thickness, and order parameters, were not significantly altered by the presence or type of bioactive lipid, regardless of the presence of GlyT2. Our work, together with previous computational and experimental data, suggests that these acyl-inhibitors of GlyT2 inhibit the transporter by directly binding to it. However, these bioactive lipids bound to various parts of GlyT2 and did not prefer a single binding site during 4.5 μs of simulation. We postulate that the binding site is located at the solvent-exposed regions of GlyT2. Understanding the mechanism of action of these and related bioactive lipids is essential in effectively developing high-affinity GlyT2 inhibitors for the treatment of pain.

Hydrogen bond-Driven Self–Assembly between Amidinium Cations and Carboxylate Anions: A Combined Molecular Dynamics, NMR Spectroscopy, and Single Crystal X-ray Diffraction Study

Publisher: Wiley

Date: 23-05-2017

DOI: 10.1002/ASIA.201700406

Abstract: A combination of molecular dynamics (MD), NMR spectroscopy, and single crystal X-ray diffraction (SCXRD) techniques was used to probe the self-assembly of para- and meta-bis(amidinium) compounds with para-, meta-, and ortho-dicarboxylates. Good concordance was observed between the MD and experimental results. In DMSO solution, the systems form several rapidly exchanging assemblies, in part because a range of hydrogen bonding interactions is possible between the amidinium and carboxylate moieties. Upon crystallization, the majority of the systems form 1D supramolecular polymers, which are held together by short N-H⋅⋅⋅O hydrogen bonds.

Cytosolic region of TM6 in P-glycoprotein: topographical analysis and functional perturbation by site directed labeling

Publisher: American Chemical Society (ACS)

Date: 28-02-2008

DOI: 10.1021/BI7023089

Abstract: Reduced intracellular drug accumulation due to the activity of the drug efflux pump ABC (B1) is a major mechanism in the resistance of cancer cells to chemotherapy. ABC (B1) is a poly specific transporter, and the molecular mechanism of its complex translocation process remains to be elucidated. To understand the process will require information on the regions involved in drug binding and those that couple this event to nucleotide hydrolysis. The present investigation focuses on the cytosolic region of transmembrane helix 6 (TM6), which has been widely attributed with a central role in the translocation process. A series of ABC (B1) isoforms containing a unique cysteine within TM6 was constructed and the resultant proteins purified and reconstituted. Accessibility of the cysteines to covalent modification by maleimide reagents was measured for the basal, ATP bound and vanadate trapped conformations of each isoform. Residues at the two extremes of the TM6 region examined (amino acids 344 to 360) were considerably more accessible than the central segment, the latter of which also failed to undergo significant conformational changes during the catalytic cycle. Covalent modification of the cytosolic segment of TM6 did, however, attenuate drug stimulation of ATP hydrolysis and demonstrates an important role for this segment in coupling drug binding to ATP hydrolysis during translocation.

ATP-binding cassette transporters in Escherichia coli

Publisher: Elsevier BV

Date: 09-2008

DOI: 10.1016/J.BBAMEM.2008.06.009

Abstract: ATP-binding cassette (ABC) transporters are integral membrane proteins that actively transport molecules across cell membranes. In Escherichia coli they consist primarily of import systems that involve in addition to the ABC transporter itself a substrate binding protein and outer membrane receptors or porins, and a number of transporters with varied functions. Recent crystal structures of a number of ATPase domains, substrate binding proteins, and full-length transporters have given new insight in the molecular basis of transport. Bioinformatics approaches allow an approximate identification of all ABC transporters in E. coli and their relation to other known transporters. Computational approaches involving modeling and simulation are beginning to yield insight into the dynamics of the transporters. We summarize the function of the known ABC transporters in E. coli and mechanistic insights from structural and computational studies.

Functional MRI Investigation of Audiovisual Interactions in Auditory Midbrain

Publisher: IEEE

Date: 07-2018

DOI: 10.1109/EMBC.2018.8513629

Homology model of the GABA(A) receptor examined using Brownian dynamics

Publisher: Elsevier BV

Date: 05-2005

DOI: 10.1529/BIOPHYSJ.104.051664

The Effect of Environment on the Recognition and Binding of Vancomycin to Native and Resistant Forms of Lipid II

Publisher: Elsevier BV

Date: 12-2011

DOI: 10.1016/J.BPJ.2011.10.047

Dysregulation of transition metal ion homeostasis is the molecular basis for cadmium toxicity in Streptococcus pneumoniae

Publisher: Springer Science and Business Media LLC

Date: 03-03-2015

DOI: 10.1038/NCOMMS7418

Abstract: Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae , occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress.

Heteroleptic Tripalladium(II) Cages

Publisher: Wiley

Date: 21-02-2022

DOI: 10.1002/ASIA.202200093

Abstract: There is a concerted attempt to develop self‐assembled metallo‐cages of greater structural complexity, and heteroleptic Pd II cages are emerging as prime candidates in these efforts. Most of these are dinuclear: few ex les of higher nuclearity have been reported. We demonstrate here a robust method for the formation of tripalladium(II) cages from the 2 : 3 : 3 combination of a tritopic ligand, Pd II , and a selection of ditopic ligands of the correct size and geometry.

Protonophoric and mitochondrial uncoupling activity of aryl-carbamate substituted fatty acids

Publisher: Royal Society of Chemistry (RSC)

Date: 2023

DOI: 10.1039/D2OB02049A

Abstract: Aryl-carbamate substituted fatty acids are protonophores that uncouple mitochondrial oxidative phosphorylation. The proton transport cycle requires self-assembly into membrane permeable dimers. The findings demonstrate the anion transport capability of the carbamate group.

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.BBAMEM.2018.07.012

Abstract: Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental s les when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1-2.0 mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50 mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10 mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20 mol% cholesterol not only modulated the fluidity of the bilayers it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.

Mutation R356Q in the collybistin phosphoinositide binding site associated with mild intellectual disability.

Publisher: Frontiers Media SA

Date: 12-03-2019

DOI: 10.3389/FNMOL.2019.00060

Visible‐Light‐Reactive Single‐Chain Nanoparticles

Publisher: Wiley

Date: 26-04-2023

DOI: 10.1002/ANIE.202302995

Abstract: We introduce a single‐chain nanoparticle (SCNP) system capable of catalyzing the photooxidation of nonpolar alkenes up to three times more efficiently than an equivalent small‐molecule photosensitizer at an identical concentration. Specifically, we construct a polymer chain constituted of poly(ethylene glycol) methyl ether methacrylate and glycidyl methacrylate which we compact via multifunctional thiol‐epoxide ligation and functionalize with Rose Bengal ( RB ) in a one pot reaction, affording SCNPs with a hydrophilic shell and hydrophobic photocatalytic regions. Photooxidation of the internal alkene in oleic acid proceeds under green light. RB confined within the SCNP is three times more effective for nonpolar alkenes than free RB in solution, which we hypothesize is due to the spatial proximity of the photosensitizing units to the substrate in the hydrophobic region. Our approach demonstrates that SCNP based catalysts can afford enhanced photocatalysis via confinement effects in a homogeneous reaction environment.

Mechanism and putative structure of B0-like neutral amino acid transporters

Publisher: Springer Science and Business Media LLC

Date: 09-2006

DOI: 10.1007/S00232-006-0879-3

Abstract: The Na(+)-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by B(0)-type neutral amino acid transporters. Two B(0)-type amino acid transporters have been identified in the neurotransmitter transporter family SLC6, namely B(0)AT1 (SLC6A19) and B(0)AT2 (SLC6A15). In contrast to other members of this family, B(0)-like transporters are chloride-independent. B(0)AT1 and B(0)AT2 preferentially bind the substrate prior to the Na(+)-ion. The Na(+)-concentration affects the K ( m ) of the substrate and vice versa. A kinetic scheme is proposed that is consistent with the experimental data. An overlapping binding site of substrate and cosubstrate has been demonstrated in the bacterial orthologue LeuT( Aa ) from Aquifex aeolicus, which elegantly explains the mutual effect of substrate and cosubstrate on each other's K ( m )-value. LeuT( Aa ) is sequence-related to transporters of the SLC6 family, allowing homology modeling of B(0)-like transporters along its structure.

A Unique Sequence Is Essential for Efficient Multidrug Efflux Function of the MtrD Protein of Neisseria gonorrhoeae

Publisher: American Society for Microbiology

Date: 31-08-2021

DOI: 10.1128/MBIO.01675-21

Abstract: The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance.

A multifunctional surfactant catalyst inspired by hydrolases

Publisher: American Association for the Advancement of Science (AAAS)

Date: 03-04-2020

DOI: 10.1126/SCIADV.AAZ0404

Abstract: Enzyme-inspired catalysis using self-assembled hiphiles mimic and localize multiple chemical units common to hydrolases.

Molecular Determinants for Substrate Interactions with the Glycine Transporter GlyT2

Publisher: American Chemical Society (ACS)

Date: 09-11-2018

DOI: 10.1021/ACSCHEMNEURO.7B00407

Abstract: Transporters in the SLC6 family play key roles in regulating neurotransmission and are the targets for a wide range of therapeutics. Important insights into the transport mechanisms and the specificity of drug interactions of SLC6 transporters have been obtained from the crystal structures of a bacterial homologue of the family, LeuT

Pore structure controls stability and molecular flux in engineered protein cages

Publisher: Cold Spring Harbor Laboratory

Date: 27-01-2021

DOI: 10.1101/2021.01.27.428512

Abstract: Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have recently been featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern cage stability and molecular flux through their pores. In this work, we systematically designed a 24-member library of protein cage variants based on the T. maritima encapsulin, each featuring pores of different size and charge. Twelve encapsulin pore variants were successfully assembled and purified, including eight designs with exceptional and prolonged thermal stability. While pores lined with negatively charged residues resulted in more robust assemblies than their corresponding positively charged variants, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures of pore variants elucidated at resolutions between 2.5-3.6 Å. Alongside these structures, molecular dynamics simulations and stopped flow kinetics experiments reveal the importance of considering both pore size and surface charge, together with flexibility and rate determining steps, when designing protein cages for controlling molecular flux.

Taxonomical considerations and molecular phylogeny of the closely related genera Bitylenchus, Sauertylenchus and Tylenchorhynchus (Nematoda: Telotylenchinae), with one new and four known species from Iran

Publisher: Cambridge University Press (CUP)

Date: 2020

DOI: 10.1017/S0022149X20000784

Abstract: During several nematological surveys in cultivated and natural habitats in Khuzestan and Zanjan provinces of Iran, a new species, Bitylenchus parvulus n. sp., two new records for Iran – namely, Tylenchorhynchus agri and Tylenchorhynchus graciliformis – and a population of Bitylenchus parvus and one of Sauertylenchus maximus were recovered and characterized based upon morphological and molecular approaches. The new species is characterized by lip region with five to seven annuli, stylet 17.7 (17.0–18.5) μm long, sub-cylindrical tail narrowing abruptly near terminus giving a bluntly digitate shape to the tail tip, cuticle near anterior part of vulva wrinkled and post-rectal sac occupies whole of tail cavity. The phylogenetic analyses were carried out using molecular data from D2–D3 expansion segments of large ribosomal subunit (28S rRNA) for all studied species and the partial small ribosomal subunit (18S rRNA) for the new species. The representatives of Bitylenchus and Sauertylenchus formed distinct clades from Tylenchorhynchus members, supporting the hypothesis in which Bitylenchus and Sauertylenchus could be considered as valid genera, but rejecting the ‘large-genus’ concept for Tylenchorhynchus . Also, Sauertylenchus ibericus was proposed as a junior synonym of S. maximus based on the results from morphological and phylogenetic analysis. Furthermore, an identification key for all known species included in the three genera Bitylenchus , Tylenchorhynchus and Sauertylenchus is presented herein. The number of transverse annuli on the lip region and presence/absence of post-rectal sac were considered as the main diagnostic characters for classifying the species into seven groups, and other morphological and morphometric characters were subsequently used for distinguishing species in each group.

Extracellular vesicle lipids in cancer immunoevasion

Publisher: Elsevier BV

Date: 11-2023

DOI: 10.1016/J.TRECAN.2023.08.006

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism

Publisher: American Chemical Society (ACS)

Date: 13-05-2021

DOI: 10.1021/ACSINFECDIS.1C00149

Abstract: Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium

The Impact of Antimicrobial Peptides on the Acinetobacter baumannii Inner Membrane Is Modulated by Lipid Polyunsaturation

Publisher: American Chemical Society (ACS)

Date: 15-03-2023

DOI: 10.1021/ACSINFECDIS.2C00530

P-glycoprotein models of the apo and ATP-bound states based on homology with Sav1866 and MalK

Publisher: Wiley

Date: 07-08-2007

DOI: 10.1016/J.FEBSLET.2007.07.069

Abstract: We exploit the biochemical and sequence similarity between Staphylococcus aureus Sav1866 and P-glycoprotein to develop a homology model of P-glycoprotein representing an ATP-bound state, which captures the major features of the low-resolution EM structure and is consistent with cysteine mutagenesis studies. Using insights from the MalK crystal structures and BtuCD simulations, we model two nucleotide-free conformations. Conformational changes are characterized by pincering rigid-body rotations of the nucleotide-binding domains, inducing transmembrane domain reorganizations which correspond to the two lowest frequency normal modes of the protein. These conformations (see supplementary material) may characterize some of the major steps in the nucleotide catalytic cycle.

The role of plasmalogens, Forssman lipids, and sphingolipid hydroxylation in modulating the biophysical properties of the epithelial plasma membrane

Publisher: AIP Publishing

Date: 03-2021

DOI: 10.1063/5.0040887

Abstract: A coarse-grain model of the epithelial plasma membrane was developed from high-resolution lipidomic data and simulated using the MARTINI force field to characterize its biophysical properties. Plasmalogen lipids, Forssman glycosphingolipids, and hydroxylated Forssman glycosphingolipids and sphingomyelin were systematically added to determine their structural effects. Plasmalogen lipids have a minimal effect on the overall biophysical properties of the epithelial plasma membrane. In line with the hypothesized role of Forssman lipids in the epithelial apical membrane, the introduction of Forssman lipids initiates the formation of glycosphingolipid-rich nanoscale lipid domains, which also include phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol (CHOL). This decreases the lateral diffusion in the extracellular leaflet, as well as the area per lipid of domain forming lipids, most notably PE. Finally, hydroxylation of the Forssman glycosphingolipids and sphingomyelin further modulates the lateral organization of the membrane. Through comparison to the previously studied average and neuronal plasma membranes, the impact of membrane lipid composition on membrane properties was characterized. Overall, this study furthers our understanding of the biophysical properties of complex membranes and the impact of lipid ersity in modulating membrane properties.

Computer simulations of ABC transporter components

Publisher: Canadian Science Publishing

Date: 12-2006

DOI: 10.1139/O06-182

Abstract: Current computer simulation techniques provide robust tools for studying the detailed structure and functional dynamics of proteins, as well as their interaction with each other and with other biomolecules. In this minireview, we provide an illustration of recent progress and future challenges in computer modeling by discussing computational studies of ATP-binding cassette (ABC) transporters. ABC transporters have multiple components that work in a well coordinated fashion to enable active transport across membranes. The mechanism by which members of this superfamily execute transport remains largely unknown. Molecular dynamics simulations initiated from high-resolution crystal structures of several ABC transporters have proven to be useful in the investigation of the nature of conformational coupling events that may drive transport. In addition, fruitful efforts have been made to predict unknown structures of medically relevant ABC transporters, such as P-glycoprotein, using homology-based computational methods. The various techniques described here are also applicable to gaining an atomically detailed understanding of the functional mechanisms of proteins in general.

Fatigue behaviour of load-bearing polymeric bone scaffolds: A review

Publisher: Elsevier BV

Date: 10-2023

DOI: 10.1016/J.ACTBIO.2023.09.048

Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding

Publisher: Proceedings of the National Academy of Sciences

Date: 13-02-2017

DOI: 10.1073/PNAS.1612927114

Abstract: At this time, multidrug-resistant gram-negative bacteria are estimated to cause approximately 700,000 deaths per year globally, with a prediction that this figure could reach 10 million a year by 2050. Antivirulence therapy, in which virulence mechanisms of a pathogen are chemically inactivated, represents a promising approach to the development of treatment options. The family of lipid A phosphoethanolamine transferases in gram-negative bacteria confers bacterial resistance to innate immune defensins and colistin antibiotics. The development of inhibitors to block lipid A phosphoethanolamine transferase could improve innate immune clearance and extend the usefulness of colistin antibiotics. The solved crystal structure and biophysical studies suggest that the enzyme undergoes large conformational changes to enable binding and catalysis of two very differently sized substrates.

Lipid fingerprints are similar between SLC6 transporters in the neuronal membrane

Publisher: Cold Spring Harbor Laboratory

Date: 21-01-2021

DOI: 10.1101/2021.01.20.427530

Abstract: We use molecular dynamics simulations to characterise the local lipid annulus, or “fingerprint”, of three SLC6 transporters (dDAT, hSERT, and GlyT2) embedded into a complex neuronal membrane. New membrane analysis tools were created to improve leaflet detection and leaflet-dependent properties. Overall, lipid fingerprints are comprised of similar lipids when grouped by headgroup or tail saturation. The enrichment and depletion of specific lipids, including sites of cholesterol contacts, varies between transporters. The subtle differences in lipid fingerprints results in varying membrane biophysical properties near the transporter. Through comparisons to previous literature, we highlight that the lipid-fingerprint in complex membranes is highly dependent on membrane composition. Furthermore, through embedding these transporters in a simplified model membrane, we show that the simplified membrane is not able to capture the biophysical properties of the complex membrane. Our results further characterise how the presence and identity of membrane proteins affects the complex interplay of lipid-protein interactions, including the local lipid environment and membrane biophysical properties. Lipid fingerprints are comprised of similar lipid classes Sites of specific lipid contacts, including CHOL, varies between transporters Changes in lipid annulus result in variable local membrane biophysical properties Membrane composition, including that of complex membranes, affects lipid annulus

The ryanodine receptor store-sensing gate controls Ca2+ waves and Ca2+-triggered arrhythmias

Publisher: Springer Science and Business Media LLC

Date: 19-01-2014

DOI: 10.1038/NM.3440

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Publisher: American Society for Microbiology

Date: 29-06-2021

DOI: 10.1128/MBIO.01070-21

Abstract: Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options.

Comparing Non-Bonded Metal Ion Models in the Divalent Cation Binding Protein PsaA

Publisher: American Chemical Society (ACS)

Date: 14-02-2020

DOI: 10.1021/ACS.JCTC.9B01180

The mechanism of ABC transporters: general lessons from structural and functional studies of an antigenic peptide transporter

Publisher: Wiley

Date: 27-01-2009

DOI: 10.1096/FJ.08-121855

Abstract: The shuttling of substrates across a cellular membrane frequently requires a specialized ATP-binding cassette (ABC) transporter, which couples the energy of ATP binding and hydrolysis to substrate transport. Due to its importance in immunity, the ABC transporter associated with antigen processing (TAP) has been studied extensively and is an excellent model for other ABC transporters. The TAP protein pumps cytosolic peptides into the endoplasmic reticulum for loading onto class I major histocompatibility complex (MHC) for subsequent immune surveillance. Here, we outline a potential mechanism for the TAP protein with supporting evidence from bacterial transporter structures. The similarities and differences between TAP and other transporters support the notion that ABC transporters in general have adapted around a universal transport mechanism.

A model of the glycine receptor deduced from Brownian dynamics studies

Publisher: Proceedings of the National Academy of Sciences

Date: 20-03-2003

DOI: 10.1073/PNAS.0630652100

Abstract: We have developed a three-dimensional model of the α1 homomeric glycine receptor by using Brownian dynamics simulations to account for its observed physiological properties. The model channel contains a large external vestibule and a shallow internal vestibule, connected by a narrow, cylindrical selectivity filter. Three rings of charged residues from the pore-lining M2 domain are modeled as point charges in the protein. Our simulations reproduce many of the key features of the channel, such as the current–voltage profiles, permeability ratios, and ion selectivity. When we replace the ring of alanine residues lining the selectivity filter with glutamates, the mutant model channel becomes permeable to cations, as observed experimentally. In this mutation, anions act as chaperones for sodium ions in the extracellular vestibule, and together they penetrate deep inside the channel against a steep energy barrier encountered by unaccompanied ions. Two subsequent amino acid mutations increase the cation permeability, enabling monovalent cations to permeate through the channel unaided and alent cations to permeate when chaperoned by anions. These results illustrate the key structural features and underlying mechanism for charge selectivity in the glycine receptor.

Molecular basis for the interaction of the mammalian amino acid transporters B0AT1 and B0AT3 with their ancillary protein collectrin

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1074/JBC.M115.648519

The reliability of molecular dynamics simulations of the multidrug transporter P-glycoprotein in a membrane environment

Publisher: Public Library of Science (PLoS)

Date: 25-01-2018

DOI: 10.1371/JOURNAL.PONE.0191882

Orientation of μ-conotoxin PIIIA in a sodium channel vestibule, based on voltage dependence of its binding

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 26-04-2011

DOI: 10.1124/MOL.111.071779

Abstract: Mutant cycle analysis has been used in previous studies to constrain possible docking orientations for various toxins. As an independent test of the bound orientation of μ-conotoxin PIIIA, a selectively targeted sodium channel pore blocker, we determined the contributions to binding voltage dependence of specific residues on the surface of the toxin. A change in the "apparent valence" (zδ) of the block, which is associated with a change of a specific toxin charge, reflects a change in the charge movement within the transmembrane electric field as the toxin binds. Toxin derivatives with charge-conserving mutations (R12K, R14K, and K17R) showed zδ values similar to those of wild type (0.61 ± 0.01, mean ± S.E.M.). Charge-changing mutations produced a range of responses. Neutralizing substitutions for Arg14 and Lys17 showed the largest reductions in zδ values, to 0.18 ± 0.06 and 0.20 ± 0.06, respectively, whereas unit charge-changing substitutions for Arg12, Ser13, and Arg20 gave intermediate values (0.24 ± 0.07, 0.33 ± 0.04, and 0.32 ± 0.05), which suggests that each of these residues contributes to the dependence of binding on the transmembrane voltage. Two mutations, R2A and G6K, yielded no significant change in zδ. These observations suggest that the toxin binds with Arg2 and Gly6 facing the extracellular solution, and Arg14 and Lys17 positioned most deeply in the pore. In this study, we used molecular dynamics to simulate toxin docking and performed Poisson-Boltzmann calculations to estimate the changes in local electrostatic potential when in idual charges were substituted on the toxin's surface. Consideration of two limiting possibilities suggests that most of the charge movement associated with toxin binding reflects sodium redistribution within the narrow part of the pore.

Lipid-mediated antimicrobial resistance: a phantom menace or a new hope?

Publisher: Springer Science and Business Media LLC

Date: 02-2022

DOI: 10.1007/S12551-021-00912-8

Abstract: The proposition of a post-antimicrobial era is all the more realistic with the continued rise of antimicrobial resistance. The development of new antimicrobials is failing to counter the ever-increasing rates of bacterial antimicrobial resistance. This necessitates novel antimicrobials and drug targets. The bacterial cell membrane is an essential and highly conserved cellular component in bacteria and acts as the primary barrier for entry of antimicrobials into the cell. Although previously under-exploited as an antimicrobial target, the bacterial cell membrane is attractive for the development of novel antimicrobials due to its importance in pathogen viability. Bacterial cell membranes are erse assemblies of macromolecules built around a central lipid bilayer core. This lipid bilayer governs the overall membrane biophysical properties and function of its membrane-embedded proteins. This mini-review will outline the mechanisms by which the bacterial membrane causes and controls resistance, with a focus on alterations in the membrane lipid composition, chemical modification of constituent lipids, and the efflux of antimicrobials by membrane-embedded efflux systems. Thorough insight into the interplay between membrane-active antimicrobials and lipid-mediated resistance is needed to enable the rational development of new antimicrobials. In particular, the union of computational approaches and experimental techniques for the development of innovative and efficacious membrane-active antimicrobials is explored.

Erratum to “DNA detection of a temporary and original user of an office space” [Forensic Sci. Int. Genet. (2020) 102203]

Publisher: Elsevier BV

Date: 07-2020

DOI: 10.1016/J.FSIGEN.2020.102277

Structural arrangement of the transmission interface in the antigen ABC transport complex TAP

Publisher: Proceedings of the National Academy of Sciences

Date: 07-04-2009

DOI: 10.1073/PNAS.0811260106

Abstract: The transporter associated with antigen processing (TAP) represents a focal point in the immune recognition of virally or malignantly transformed cells by translocating proteasomal degradation products into the endoplasmic reticulum–lumen for loading of MHC class I molecules. Based on a number of experimental data and the homology to the bacterial ABC exporter Sav1866, we constructed a 3D structural model of the core TAP complex and used it to examine the interface between the transmembrane and nucleotide-binding domains (NBD) by cysteine-scanning and cross-linking approaches. Herein, we demonstrate the functional importance of the newly identified X-loop in the NBD in coupling substrate binding to downstream events in the transport cycle. We further verified domain swapping in a heterodimeric ABC half-transporter complex by cysteine cross-linking. Strikingly, either substrate binding or translocation can be blocked by cross-linking the X-loop to coupling helix 2 or 1, respectively. These results resolve the structural arrangement of the transmission interface and point to different functions of the cytosolic loops and coupling helices in substrate binding, signaling, and transport.

The Effect of Environment on the Structure of a Membrane Protein: P-Glycoprotein under Physiological Conditions

Publisher: American Chemical Society (ACS)

Date: 11-06-2012

DOI: 10.1021/CT300254Y

Abstract: The stability of the crystal structure of the multidrug transporter P-glycoprotein proposed by Aller et al. (PDBid 3G5U ) has been examined under different environmental conditions using molecular dynamics. We show that in the presence of the detergent cholate, the structure of P-glycoprotein solved at pH 7.5 is stable. However, when incorporated into a cholesterol-enriched POPC membrane in the presence of 150 mM NaCl, the structure rapidly deforms. Only when the simulation conditions closely matched the experimental conditions under which P-glycoprotein is transport active was a stable conformation obtained. Specifically, the presence of Mg(2+), which bound to distinct sites in the nucleotide binding domains (NBDs), and the double protonation of the catalytic histidines (His583 and His1228) and His149 were required. While the structure obtained in a membrane environment under these conditions is very similar to the crystal structure of Aller et al., there are several key differences. The NBDs are in direct contact, reminiscent of the open state of MalK. The angle between the transmembrane domains is also increased, resulting in an outward motion of the intracellular loops. Notably, the structures obtained from the simulations provide a better match to a range of experimental cross-linking data than does the original 3G5U-a crystal structure. This work highlights the effect small changes in environmental conditions can have of the conformation of a membrane protein and the importance of representing the experimental conditions appropriately in modeling studies.

Hydrophobic Shielding Drives Catalysis of Hydride Transfer in a Family of F420H2-Dependent Enzymes

Publisher: American Chemical Society (ACS)

Date: 30-11-2016

DOI: 10.1021/ACS.BIOCHEM.6B00683

Abstract: A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, we have used a combination of quantum mechanics and molecular dynamics calculations to study the catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis. The preferred pathway involves an initial hydride transfer step from the deprotonated cofactor (i.e., F

Environmental enrichment leads to behavioral circadian shifts enhancing brain-wide functional connectivity between sensory cortices and eliciting increased hippocampal spiking

Publisher: Elsevier BV

Date: 05-2022

DOI: 10.1016/J.NEUROIMAGE.2022.119016

Abstract: Environmental enrichment induces widespread neuronal changes, but the initiation of the cascade is unknown. We ascertained the critical period of ergence between environmental enriched (EE) and standard environment (SE) mice using continuous infrared (IR) videography, functional magnetic resonance imaging (fMRI), and neuron level calcium imaging. Naïve adult male mice (n = 285, C57BL/6J, postnatal day 60) were ided into SE and EE groups. We assessed the linear time-series of motion activity using a novel structural break test which examined the dataset for change in circadian and day-by-day motion activity. fMRI was used to map brain-wide response using a functional connectome analysis pipeline. Awake calcium imaging was performed on the dorsal CA1 pyramidal layer. We found the preeminent behavioral feature in EE was a forward shift in the circadian rhythm, prolongation of activity in the dark photoperiod, and overall decreased motion activity. The crepuscular period of dusk was seen as the critical period of ergence between EE and SE mice. The functional processes at dusk in EE included increased functional connectivity in the visual cortex, motor cortex, retrosplenial granular cortex, and cingulate cortex using seed-based analysis. Network based statistics found a modulated functional connectome in EE concentrated in two hubs: the hippoc al formation and isocortical network. These hubs experienced a higher node degree and significant enhanced edge connectivity. Calcium imaging revealed increased spikes per second and maximum firing rate in the dorsal CA1 pyramidal layer, in addition to location (anterior-posterior and medial-lateral) effect size differences between EE and SE. The emergence of functional-neuronal changes due to enrichment consisted of enhanced hippoc al-isocortex functional connectivity and CA1 neuronal increased spiking linked to a circadian shift during the dusk period. Future studies should explore the molecular consequences of enrichment inducing shifts in the circadian period.

Conduction Mechanisms of Chloride Ions in ClC-Type Channels

Publisher: Elsevier BV

Date: 02-2004

DOI: 10.1016/S0006-3495(04)74160-0

Rangefinder: A Semisynthetic FRET Sensor Design Algorithm

Publisher: American Chemical Society (ACS)

Date: 15-11-2016

DOI: 10.1021/ACSSENSORS.6B00576

Effect of the Force Field on Molecular Dynamics Simulations of the Multidrug Efflux Protein P-Glycoprotein

Publisher: American Chemical Society (ACS)

Date: 10-09-2021

DOI: 10.1021/ACS.JCTC.1C00414

Abstract: Molecular dynamics (MD) simulations have been used extensively to study P-glycoprotein (P-gp), a flexible multidrug transporter that is a key player in the development of multidrug resistance to chemotherapeutics. A substantial body of literature has grown from simulation studies that have employed various simulation conditions and parameters, including AMBER, CHARMM, OPLS, GROMOS, and coarse-grained force fields, drawing conclusions from simulations spanning hundreds of nanoseconds. Each force field is typically parametrized and validated on different data and observables, usually of small molecules and peptides there have been few comparisons of force field performance on large protein-membrane systems. Here we compare the conformational ensembles of P-gp embedded in a POPC/cholesterol bilayer generated over 500 ns of replicate simulation with five force fields from popular biomolecular families: AMBER 99SB-ILDN, CHARMM 36, OPLS-AA/L, GROMOS 54A7, and MARTINI. We find considerable differences among the ensembles with little conformational overlap, although they correspond to similar extents to structural data obtained from electron paramagnetic resonance and cross-linking studies. Moreover, each trajectory was still s ling new conformations at a high rate after 500 ns of simulation, suggesting the need for more s ling. This work highlights the need to consider known limitations of the force field used (e.g., biases toward certain secondary structures) and the simulation itself (e.g., whether sufficient s ling has been achieved) when interpreting accumulated results of simulation studies of P-gp and other transport proteins.

Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2

Publisher: Life Science Alliance, LLC

Date: 23-01-2023

DOI: 10.26508/LSA.202201708

Abstract: Membrane cholesterol binds to and modulates the function of various SLC6 neurotransmitter transporters, including stabilizing the outward-facing conformation of the dopamine and serotonin transporters. Here, we investigate how cholesterol binds to GlyT2 (SLC6A5), modulates glycine transport rate, and influences bioactive lipid inhibition of GlyT2. Bioactive lipid inhibitors are analgesics that bind to an allosteric site accessible from the extracellular solution when GlyT2 adopts an outward-facing conformation. Using molecular dynamics simulations, mutagenesis, and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a binding site formed by transmembrane helices 1, 5, and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3′ hydroxyl group into the cholesterol binding cavity, close to the allosteric site. The synergy between cholesterol and allosteric inhibitors provides a novel mechanism of inhibition and a potential avenue for the development of potent GlyT2 inhibitors as alternative therapeutics for the treatment of neuropathic pain and therapeutics that target other SLC6 transporters.

The inhibitory activity of glycine transporter 2 targeting bioactive lipid analgesics are influenced by formation of a deep lipid cavity

Publisher: Cold Spring Harbor Laboratory

Date: 10-09-2020

DOI: 10.1101/2020.09.10.290908

Abstract: The human glycine transporter 2 (GlyT2 or SLC6A5) has emerged as a promising drug target for the development of new analgesics to manage chronic pain. N-acyl amino acids inhibit GlyT2 through binding to an allosteric binding site to produce analgesia in vivo with minimal overt side effects. In this paper we use a combination of medicinal chemistry, electrophysiology, and computational modelling to explore the molecular basis of GlyT2 inhibition at the allosteric site. We show how N-acyl amino acid head group stereochemistry, tail length and double bond position promote enhanced inhibition by deep penetration into the binding pocket. This work provides new insights into the interaction of lipids with transport proteins and will aid in future rational design of novel GlyT2 inhibitors.

Structure-based interpretation of the mutagenesis database for the nucleotide binding domains of P-glycoprotein

Publisher: Elsevier BV

Date: 02-2008

DOI: 10.1016/J.BBAMEM.2007.10.021

Abstract: P-glycoprotein (P-gp) is the most intensively studied eukaryotic ATP binding cassette (ABC) transporter, due to its involvement in the multidrug resistance phenotype of a number of cancers. In common with most ABC transporters, P-gp is comprised of two transmembrane domains (TMDs) and two nucleotide binding domains (NBD), the latter coupling ATP hydrolysis with substrate transport (efflux in the case of P-gp). Biochemical investigations over the past twenty years have attempted to unlock mechanistic aspects of P-glycoprotein through scanning and site-directed mutagenesis of both the TMDs and the NBDs. Contemporaneously, crystallographers have elucidated the atomic structure of numerous ABC transporter NBDs, as well as the intact structure (i.e. NBDs and TMDs) of a distantly related ABC-exporter Sav1866. Significantly, the structure of P-gp remains unknown, and only low resolution electron microscopy data exists. Within the current manuscript we employ crystallographic data for homologous proteins, and a molecular model for P-gp, to perform a structural interpretation of the existing "mutagenesis database" for P-gp NBDs. Consequently, this will enable testable predictions to be made that will result in further in-roads into our understanding of this clinically important drug pump.

Pore structure controls stability and molecular flux in engineered protein cages

Publisher: American Association for the Advancement of Science (AAAS)

Date: 04-02-2022

DOI: 10.1126/SCIADV.ABL7346

Abstract: Protein cages are a common architectural motif used by living organisms to compartmentalize and control biochemical reactions. While engineered protein cages have featured in the construction of nanoreactors and synthetic organelles, relatively little is known about the underlying molecular parameters that govern stability and flux through their pores. In this work, we systematically designed 24 variants of the Thermotoga maritima encapsulin cage, featuring pores of different sizes and charges. Twelve pore variants were successfully assembled and purified, including eight designs with exceptional thermal stability. While negatively charged mutations were better tolerated, we were able to form stable assemblies covering a full range of pore sizes and charges, as observed in seven new cryo-EM structures at 2.5- to 3.6-Å resolution. Molecular dynamics simulations and stopped-flow experiments revealed the importance of considering both pore size and charge, together with flexibility and rate-determining steps, when designing protein cages for controlling molecular flux.

Is protein structure enough? A review of the role of lipids in SLC6 transporter function

Publisher: Elsevier BV

Date: 05-2019

DOI: 10.1016/J.NEULET.2018.05.020

Abstract: SLC6 neurotransmitter transporters facilitate the Na

Permeation dynamics of chloride ions in the CIC-0 and CIC-1 channels

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.CPLETT.2004.01.072

Vancomycin: ligand recognition, dimerization and super-complex formation

Publisher: Wiley

Date: 11-02-2013

DOI: 10.1111/FEBS.12121

Abstract: The antibiotic vancomycin targets lipid II, blocking cell wall synthesis in Gram-positive bacteria. Despite extensive study, questions remain regarding how it recognizes its primary ligand and what is the most biologically relevant form of vancomycin. In this study, molecular dynamics simulation techniques have been used to examine the process of ligand binding and dimerization of vancomycin. Starting from one or more vancomycin monomers in solution, together with different peptide ligands derived from lipid II, the simulations predict the structures of the ligated monomeric and dimeric complexes to within 0.1 nm rmsd of the structures determined experimentally. The simulations reproduce the conformation transitions observed by NMR and suggest that proposed differences between the crystal structure and the solution structure are an artifact of the way the NMR data has been interpreted in terms of a structural model. The spontaneous formation of both back-to-back and face-to-face dimers was observed in the simulations. This has allowed a detailed analysis of the origin of the cooperatively between ligand binding and dimerization and suggests that the formation of face-to-face dimers could be functionally significant. The work also highlights the possible role of structural water in stabilizing the vancomycin ligand complex and its role in the manifestation of vancomycin resistance.

PsiRESP: calculating RESP charges with Psi4

Publisher: The Open Journal

Date: 04-05-2022

DOI: 10.21105/JOSS.04100

Capturing the Dynamics of a Spring-Loaded Protein

Publisher: Elsevier BV

Date: 07-2017

DOI: 10.1016/J.STR.2017.06.008

Abstract: Skp and other holdase chaperones bind unfolded bacterial outer membrane proteins, preventing premature folding until they insert into the membrane. In this issue of Structure, Holdbrook et al. (2017) use a combination of NMR, SAXS, ensemble optimization, and MD simulations to show that the Skp chaperone s les a much wider range of conformations than suggested by its structure alone.

Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein

Publisher: Elsevier BV

Date: 2017

DOI: 10.1016/J.BCP.2016.10.002

Abstract: The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity.

Structural Characterization of Two Metastable ATP-Bound States of P-Glycoprotein

Publisher: Public Library of Science (PLoS)

Date: 14-03-2014

DOI: 10.1371/JOURNAL.PONE.0091916

Cross-linking, DEER-spectroscopy and molecular dynamics confirm the inward facing state of P-glycoprotein in a lipid membrane

Publisher: Elsevier BV

Date: 07-2020

DOI: 10.1016/J.JSB.2020.107513

Intestinal peptidases form functional complexes with the neutral amino acid transporter B⁰AT1

Publisher: Portland Press Ltd.

Date: 27-07-2012

DOI: 10.1042/BJ20120307

Abstract: The brush-border membrane of the small intestine and kidney proximal tubule are the major sites for the absorption and re-absorption of nutrients in the body respectively. Transport of amino acids is mediated through the action of numerous secondary active transporters. In the mouse, neutral amino acids are transported by B0AT1 [broad neutral (0) amino acid transporter 1 SLC6A19 (solute carrier family 6 member 19)] in the intestine and by B0AT1 and B0AT3 (SLC6A18) in the kidney. Immunoprecipitation and Blue native electrophoresis of intestinal brush-border membrane proteins revealed that B0AT1 forms complexes with two peptidases, APN (aminopeptidase N/CD13) and ACE2 (angiotensin-converting enzyme 2). Physiological characterization of B0AT1 expressed together with these peptidases in Xenopus laevis oocytes revealed that APN increased the substrate affinity of the transporter up to 2.5-fold and also increased its surface expression (Vmax). Peptide competition experiments, in silico modelling and site-directed mutagenesis of APN suggest that the catalytic site of the peptidase is involved in the observed changes of B0AT1 apparent substrate affinity, possibly by increasing the local substrate concentration. These results provide evidence for the existence of B0AT1-containing digestive complexes in the brush-border membrane, interacting differentially with various peptidases, and responding to the dynamic needs of nutrient absorption in the intestine and kidney.

Transmembrane Helix 12 Modulates Progression of the ATP Catalytic Cycle in ABCB1

Publisher: American Chemical Society (ACS)

Date: 04-06-2009

DOI: 10.1021/BI900373X

Identification of a 3^rd Na⁺ binding site of the glycine transporter, GlyT2

Publisher: Public Library of Science (PLoS)

Date: 23-06-2016

DOI: 10.1371/JOURNAL.PONE.0157583

Transmembrane helix 12 plays a pivotal role in coupling energy provision and drug binding in ABCB1

Publisher: Wiley

Date: 20-08-2010

DOI: 10.1111/J.1742-4658.2010.07789.X

Abstract: Describing the molecular details of the multidrug efflux process of ABCB1, in particular the interdomain communication associated with bioenergetic coupling, continues to prove difficult. A number of investigations to date have implicated transmembrane helix 12 (TM12) in mediating communication between the transmembrane domains and nucleotide-binding domains (NBDs) of ABCB1. The present investigation further addressed the role of TM12 in ABCB1 by characterizing its topography during the multidrug efflux process with the use of cysteine-directed mutagenesis. Cysteines were introduced at various positions along TM12 and assessed for their ability to covalently bind thiol-reactive fluorescent probes with differing physiochemical properties. By analysing each isoform in the basal, ATP-bound and posthydrolytic states, it was possible to determine how the local environment of TM12 alters during the catalytic cycle. Labelling with hydrophobic CM and zwitterionic BM was extensive throughout the helix in the basal, prehydrolytic and posthydrolytic states, suggesting that TM12 is in a predominantly hydrophobic environment. Overall, the carboxy region (intracellular half) of TM12 appeared to be more responsive to changes in the catalytic state of the protein than the amino region (extracellular half). Thus, the carboxy region of TM12 is suggested to be responsive to nucleotide binding and hydrolysis at the NBDs and therefore directly involved in interdomain communication. This data can be reconciled with an atomic-scale model of human ABCB1. Taken together, these results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1 and, in particular, in coordinating conformational changes between the NBDs and transmembrane domains.

Thylakoid ultrastructure: visualizing the photosynthetic machinery

Publisher: Springer International Publishing

Date: 2017

DOI: 10.1007/978-3-319-66360-9_7

Understanding the Link between Lipid Diversity and the Biophysical Properties of the Neuronal Plasma Membrane

Publisher: American Chemical Society (ACS)

Date: 07-08-2020

DOI: 10.1021/ACS.BIOCHEM.0C00524

Effect of Poly(ethylene glycol) (PEG) Spacers on the Conformational Properties of Small Peptides: A Molecular Dynamics Study

Publisher: American Chemical Society (ACS)

Date: 12-2010

DOI: 10.1021/LA103800H

Abstract: Poly(ethylene glycol) (PEG) is used as an inert spacer in a wide range of biotechnological applications such as to display peptides and proteins on surfaces for diagnostic purposes. In such applications it is critical that the peptide is accessible to solvent and that the PEG does not affect the conformational properties of the peptide to which it is attached. Using molecular dynamics (MD) simulation techniques, we have investigated the influence of a commonly used PEG spacer on the conformation properties of a series of five peptides with differing physical-chemical properties (YGSLPQ, VFVVFV, GSGGSG, EEGEEG, and KKGKKG). The conformational properties of the peptides were compared (a) free in solution, (b) attached to a PEG-11 spacer in solution, and (c) constrained to a two-dimensional lattice via a (PEG-11)(3) spacer, mimicking a peptide displayed on a surface as used in microarray techniques. The simulations suggest that the PEG spacer has little effect on the conformational properties of small neutral peptides but has a significant effect on the conformational properties of small highly charged peptides. When constrained to a two-dimensional surface at peptide densities similar to those used experimentally, it was found that the peptides, in particular the polar and nonpolar peptides, aggregated strongly. The peptides also partitioned into the PEG layer. Potentially, this means that at high packing densities only a small fraction of the peptide attached to the surface would in fact be accessible to a potential interaction partner.

Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2

Publisher: Cold Spring Harbor Laboratory

Date: 03-09-2022

DOI: 10.1101/2022.08.31.506132

Abstract: Membrane cholesterol binds to and modulates the function of the specific SLC6 transporters. Here we investigate how cholesterol binds to and modulates the rate of glycine transport by the SLC6 glycine transporter GlyT2, and how this impacts lipid inhibition of GlyT2. Bioactive lipid inhibitors of GlyT2 are analgesics that bind to the lipid allosteric site of the outward facing GlyT2 conformation that is accessible from the extracellular solution. Using molecular dynamics simulations, mutagenesis and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a cholesterol binding site formed by transmembrane helices 1, 5 and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3’ hydroxyl group into the cholesterol binding cavity to interact with the base of the lipid allosteric site and the bound inhibitor. The recruitment of membrane cholesterol by allosteric GlyT2 inhibitors is a potential avenue for the development of high-potency, specific pain analgesics and could provide alternative therapeutics that target GlyT2 and other SLC6 neurotransmitter transporters.

Aryl urea substituted fatty acids: a new class of protonophoric mitochondrial uncoupler that utilises a synthetic anion transporter

Publisher: Royal Society of Chemistry (RSC)

Date: 2020

DOI: 10.1039/D0SC02777D

Abstract: A new mitochondrial uncoupler that forms membrane permeable dimers through interactions of remote acidic and anion receptor groups.

Structural basis of promiscuous substrate transport by Organic Cation Transporter 1

Publisher: Springer Science and Business Media LLC

Date: 11-10-2023

DOI: 10.1038/S41467-023-42086-9

The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis

Publisher: Wiley

Date: 04-2014

DOI: 10.1111/FEBS.12773

Loss, gain and altered function of GlyR α2 subunit mutations in neurodevelopmental disorders

Publisher: Frontiers Media SA

Date: 29-04-2022

DOI: 10.3389/FNMOL.2022.886729

Abstract: Glycine receptors (GlyRs) containing the α2 subunit govern cell fate, neuronal migration and synaptogenesis in the developing cortex and spinal cord. Rare missense variants and microdeletions in the X-linked GlyR α2 subunit gene ( GLRA2 ) have been associated with human autism spectrum disorder (ASD), where they typically cause a loss-of-function via protein truncation, reduced cell-surface trafficking and/or reduced glycine sensitivity (e.g., GLRA2 Δex8-9 and extracellular domain variants p.N109S and p.R126Q). However, the GlyR α2 missense variant p.R323L in the intracellular M3-M4 domain results in a gain-of-function characterized by slower synaptic decay times, longer duration active periods and increases in channel conductance. This study reports the functional characterization of four missense variants in GLRA2 associated with ASD or developmental disorders (p.V-22L, p.N38K, p.K213E, p.T269M) using a combination of bioinformatics, molecular dynamics simulations, cellular models of GlyR trafficking and electrophysiology in artificial synapses. The GlyR α2 V–22L variant resulted in altered predicted signal peptide cleavage and a reduction in cell-surface expression, suggestive of a partial loss-of-function . Similarly, GlyR α2 N38K homomers showed reduced cell-surface expression, a reduced affinity for glycine and a reduced magnitude of IPSCs in artificial synapses. By contrast, GlyR α2 K213E homomers showed a slight reduction in cell-surface expression, but IPSCs were larger, with faster rise/decay times, suggesting a gain-of-function . Lastly, GlyR α2 T269M homomers exhibited a high glycine sensitivity accompanied by a substantial leak current, suggestive of an altered function that could dramatically enhance glycinergic signaling. These results may explain the heterogeneity of clinical phenotypes associated with GLRA2 mutations and reveal that missense variants can result in a loss, gain or alteration of GlyR α2 function. In turn, these GlyR α2 missense variants are likely to either negatively or positively deregulate cortical progenitor homeostasis and neuronal migration in the developing brain, leading to changes in cognition, learning, and memory.

Site of Cholesterol Oxidation Impacts Its Localization and Domain Formation in the Neuronal Plasma Membrane

Publisher: American Chemical Society (ACS)

Date: 11-10-2021

DOI: 10.1021/ACSCHEMNEURO.1C00395

Simple Design of an Enzyme-Inspired Supported Catalyst Based on a Catalytic Triad

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.CHEMPR.2017.04.004

Non-linear intramolecular interactions and voltage sensitivity of a KV1 family potassium channel from Polyorchis penicillatus(Eschscholtz 1829)

Publisher: The Company of Biologists

Date: 11-2008

DOI: 10.1242/JEB.022608

Abstract: Voltage sensitivity of voltage-gated potassium channels (VKCs) is a primary factor in shaping action potentials in excitable cells. Variation in the amino acid sequence of the channel proteins is responsible for differences in the voltage range over which the channel opens. Thus, understanding how changes in voltage sensitivity are effected by changes in channel protein sequence illuminates the functional evolution of excitability. The KV1-family channel jShak1, from the jellyfish Polyorchis penicillatus, differs from most other KV1 channels in ways that are useful for studying the problem of how voltage sensitivity is related to channel sequence. We assessed the contributions of changes in sequence of the S4, voltage sensing, helix and changes in one asparagine residue in the S2 helix, to the relative stability of the open and closed states of the channel. Mutation of the neutral S2 residue (Asn227) to glutamate stabilized the open conformation of the channel. Different modifications of charge and length in S4 favoured either the closed conformation or the open conformation. The interactions between pairs of mutations revealed that some of the S4 mutations alter the conformation of the voltage-sensing domain such that the S4 helix is constrained to be closer to the S2 helix than in the wild-type conformation. These results, taken in conjunction with three-dimensional models of the channel, identify intra-molecular interactions that control the balance between open and closed states. These interactions are likely to be relevant to understanding the functional characteristics of members of this channel family from other organisms.

Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment.

Publisher: Springer New York

Date: 2017

DOI: 10.1007/978-1-4939-6940-1_6

Abstract: Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.

Identification of an allosteric binding site on the human glycine transporter, GlyT2, for bioactive lipid analgesics

Publisher: eLife Sciences Publications, Ltd

Date: 17-10-2019

DOI: 10.7554/ELIFE.47150

Abstract: The treatment of chronic pain is poorly managed by current analgesics, and there is a need for new classes of drugs. We recently developed a series of bioactive lipids that inhibit the human glycine transporter GlyT2 (SLC6A5) and provide analgesia in animal models of pain. Here, we have used functional analysis of mutant transporters combined with molecular dynamics simulations of lipid-transporter interactions to understand how these bioactive lipids interact with GlyT2. This study identifies a novel extracellular allosteric modulator site formed by a crevice between transmembrane domains 5, 7, and 8, and extracellular loop 4 of GlyT2. Knowledge of this site could be exploited further in the development of drugs to treat pain, and to identify other allosteric modulators of the SLC6 family of transporters.

Investigating the lipid fingerprint of SLC6 neurotransmitter transporters: a comparison of dDAT, hDAT, hSERT, and GlyT2

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.BBADVA.2021.100010

A potential new, stable state of the E-cadherin strand-swapped dimer in solution

Publisher: Springer Science and Business Media LLC

Date: 15-06-2018

DOI: 10.1007/S00249-017-1229-3

Abstract: E-cadherin is a transmembrane glycoprotein that facilitates inter-cellular adhesion in the epithelium. The ectodomain of the native structure is comprised of five repeated immunoglobulin-like domains. All E-cadherin crystal structures show the protein in one of three alternative conformations: a monomer, a strand-swapped trans homodimer and the so-called X-dimer, which is proposed to be a kinetic intermediate to forming the strand-swapped trans homodimer. However, previous studies have indicated that even once the trans strand-swapped dimer is formed, the complex is highly dynamic and the E-cadherin monomers may reorient relative to each other. Here, molecular dynamics simulations have been used to investigate the stability and conformational flexibility of the human E-cadherin trans strand-swapped dimer. In four independent, 100 ns simulations, the dimer moved away from the starting structure and converged to a previously unreported structure, which we call the Y-dimer. The Y-dimer was present for over 90% of the combined simulation time, suggesting that it represents a stable conformation of the E-cadherin dimer in solution. The Y-dimer conformation is stabilised by interactions present in both the trans strand-swapped dimer and X-dimer crystal structures, as well as additional interactions not found in any E-cadherin dimer crystal structures. The Y-dimer represents a previously unreported, stable conformation of the human E-cadherin trans strand-swapped dimer and suggests that the available crystal structures do not fully capture the conformations that the human E-cadherin trans homodimer adopts in solution.

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein

Publisher: American Society for Microbiology

Date: 24-12-2019

DOI: 10.1128/MBIO.02277-19

Abstract: With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae , the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

Simple synthetic route to an enzyme-inspired transesterification catalyst

Publisher: Royal Society of Chemistry (RSC)

Date: 2022

DOI: 10.1039/D2CY00744D

Abstract: Self-assembling transesterification catalyst inspired by the catalytic triad.

The structural basis of bacterial manganese import.

Publisher: American Association for the Advancement of Science (AAAS)

Date: 06-08-2021

DOI: 10.1126/SCIADV.ABG3980

Abstract: Bacterial manganese import is achieved by unique architectural features that are conserved across the kingdoms of life.

Residue G346 in transmembrane segment six is involved in inter-domain communication in P-glycoprotein

Publisher: American Chemical Society (ACS)

Date: 14-08-2007

DOI: 10.1021/BI700447P

Abstract: Multidrug transporters such as P-glycoprotein require considerable inter-domain communication to couple energy utilization with substrate translocation. Elucidation of the regions or residues involved in these communication pathways is a key step in the eventual molecular description of multidrug transport. We used cysteine-scanning mutagenesis to probe the functional involvement of residues along the cytoplasmic half of transmembrane segment 6 (TM6) and its extension toward the nucleotide binding domain. The mutation of one residue (G346C) in this segment adversely affected drug transport in cells. Further investigation using purified protein revealed that the underlying biochemical effect was a reduction in basal ATP hydrolysis. This G346C mutation also affected the stimulation of ATPase activity in a drug dependent manner but had no effect on drug binding, ATP binding, or ADP release. Homology modeling of P-glycoprotein indicated that the G346C mutation caused a steric interaction between TM5 and TM6, thereby precluding a helical movement required to support ATP hydrolysis.

Центр Научно-технических И Финансовых Исследований

Location: Kazakhstan

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School Of Agricultural Engineering And Environment- Universidad Politécnica De Valencia

Location: Spain

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Institute For Sustainable Agriculture-CSIC

Location: Spain

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Plant-Pathology (Agronomy Department)-Universidad De Córdoba

Location: Spain

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University Of Queensland

Location: Australia

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The University Of Canberra

Location: Australia

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Australian National University

Location: Australia

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Discovery Projects - Grant ID: DP200100535

Start Date: 2020

End Date: 12-2022

Amount: $420,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE190100160

Start Date: 2019

End Date: 12-2019

Amount: $368,994.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP180103573

Start Date: 2018

End Date: 12-2021

Amount: $604,543.00

Funder: Australian Research Council

Discovery Early Career Researcher Award - Grant ID: DE120101550

Start Date: 01-2012

End Date: 06-2016

Amount: $375,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP130102153

Start Date: 2013

End Date: 12-2016

Amount: $946,000.00

Funder: Australian Research Council