scholarly journals Substrate Transport is Mediated not only by P-glycoprotein but also by Lipid Penetration

2019 ◽  
Author(s):  
Yuqi Yu ◽  
Jinan Wang ◽  
Zhaoqiang Chen ◽  
Guimin Wang ◽  
Zhijian Xu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Scale ◽  
Md Simulations ◽  
Glycoprotein P ◽  
Transport Pathways ◽  
Future Design ◽  
P Glycoprotein ◽  
Complex Biological Process ◽  
Multiple Ligands ◽  
Ligand Transport

ABSTRACTIn association with large-scale conformational changes, the members of the ATP-binding cassette (ABC) transporter superfamily such as P-glycoprotein (P-gp) pump endogenous cytotoxic substances and exogenous drugs out of cells. Here, a series of nonequilibrium-driven molecular dynamics (MD) simulations are sophisticatedly combined to provide a generally effective access to quantitatively investigate such a complex biological process that has been posing a great challenge for experiments and computational simulations. Both common features and unique characteristics of multiple ligands (substrates or inhibitors) that are recognized by P-gps from mouse and human species are quantitatively explored, providing additional insights into experimentally suggested ligand transport pathways and summarizing the important roles of not only different P-gps but also lipids in regulating ligand transport. These findings reveal the molecular mechanism underlying the transport of ligands by P-gps from different species and emphasize the consideration of lipid effects on the future design of effective P-gp inhibitors.

scholarly journals Homology modelling of human P-glycoprotein

2015 ◽  
Vol 43 (5) ◽  
pp. 952-958 ◽  
Author(s):  
Laura Domicevica ◽  
Philip C. Biggin
Keyword(s):  
Structural Biology ◽  
Conformational Changes ◽  
Homology Modelling ◽  
Md Simulations ◽  
Multi Drug Resistance ◽  
Glycoprotein P ◽  
The Past ◽  
Atp Binding Cassette Transporter ◽  
P Glycoprotein ◽  
Great Progress

P-glycoprotein (P-gp) is an ATP-binding cassette transporter that exports a huge range of compounds out of cells and is thus one of the key proteins in conferring multi-drug resistance in cancer. Understanding how it achieves such a broad specificity and the series of conformational changes that allow export to occur form major, on-going, research objectives around the world. Much of our knowledge to date has been derived from mutagenesis and assay data. However, in recent years, there has also been great progress in structural biology and although the structure of human P-gp has not yet been solved, there are now a handful of related structures on which homology models can be built to aid in the interpretation of the vast amount of experimental data that currently exists. Many models for P-gp have been built with this aim, but the situation is complicated by the apparent flexibility of the system and by the fact that although many potential templates exist, there is large variation in the conformational state in which they have been crystallized. In this review, we summarize how homology modelling has been used in the past, how models are typically selected and finally illustrate how MD simulations can be used as a means to give more confidence about models that have been generated via this approach.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

scholarly journals Transport of Alzheimer’s associated amyloid-β catalyzed by P-glycoprotein

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250371
Author(s):  
James W. McCormick ◽  
Lauren Ammerman ◽  
Gang Chen ◽  
Pia D. Vogel ◽  
John G. Wise
Keyword(s):  
Cell Culture ◽  
Atp Hydrolysis ◽  
Amyloid Β ◽  
Md Simulations ◽  
Membrane Transporter ◽  
Biochemical Activity ◽  
Glycoprotein P ◽  
P Glycoprotein ◽  
Hydrolysis Activity

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimer’s disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimer’s associated amyloid-β (Aβ) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports Aβ. We observed transport of Aβ40 and Aβ42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled Aβ42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that Aβ42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

scholarly journals A Virtual Reality Ensemble Molecular Dynamics Workflow to Study Complex Conformational Changes in Proteins

2020 ◽  
Author(s):  
Jordi Juárez-Jiménez ◽  
Philip Tew ◽  
Michael o'connor ◽  
Salome Llabres ◽  
Rebecca Sage ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Virtual Reality ◽  
Conformational Changes ◽  
Large Scale ◽  
A Priori ◽  
Computational Cost ◽  
Md Simulations ◽  
Collective Variables ◽  
Sampling Protocols ◽  
State Models

<p>Molecular dynamics (MD) simulations are increasingly used to elucidate relationships between protein structure, dynamics and their biological function. Currently it is extremely challenging to perform MD simulations of large-scale structural rearrangements in proteins that occur on millisecond timescales or beyond, as this requires very significant computational resources, or the use of cumbersome ‘collective variable’ enhanced sampling protocols. Here we describe a framework that combines ensemble MD simulations and virtual-reality visualization (eMD-VR) to enable users to interactively generate realistic descriptions of large amplitude, millisecond timescale protein conformational changes in proteins. Detailed tests demonstrate that eMD-VR substantially decreases the computational cost of folding simulations of a WW domain, without the need to define collective variables <i>a priori</i>. We further show that eMD-VR generated pathways can be combined with Markov State Models to describe the thermodynamics and kinetics of large-scale loop motions in the enzyme cyclophilin A. Our results suggest eMD-VR is a powerful tool for exploring protein energy landscapes in bioengineering efforts. </p>

scholarly journals Combining Virtual Reality Visualization with Ensemble Molecular Dynamics to Study Complex Protein Conformational Changes

2020 ◽  
Author(s):  
Jordi Juárez-Jiménez ◽  
Philip Tew ◽  
Michael o'connor ◽  
Salome Llabres ◽  
Rebecca Sage ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Virtual Reality ◽  
Conformational Changes ◽  
Large Scale ◽  
Computational Cost ◽  
Md Simulations ◽  
Collective Variables ◽  
Protein Conformational Changes ◽  
Sampling Protocols ◽  
State Models

<p>Molecular dynamics (MD) simulations are increasingly used to elucidate relationships between protein structure, dynamics and their biological function. Currently it is extremely challenging to perform MD simulations of large-scale structural rearrangements in proteins that occur on millisecond timescales or beyond, as this requires very significant computational resources, or the use of cumbersome ‘collective variable’ enhanced sampling protocols. Here we describe a framework that combines ensemble MD simulations and virtual-reality visualization (eMD-VR) to enable users to interactively generate realistic descriptions of large amplitude, millisecond timescale protein conformational changes in proteins. Detailed tests demonstrate that eMD-VR substantially decreases the computational cost of folding simulations of a WW domain, without the need to define collective variables <i>a priori</i>. We further show that eMD-VR generated pathways can be combined with Markov State Models to describe the thermodynamics and kinetics of large-scale loop motions in the enzyme cyclophilin A. Our results suggest eMD-VR is a powerful tool for exploring protein energy landscapes in bioengineering efforts. </p>

Molecular Basis Explanation of Imatinib Resistance of Bcr-Abl Due to T315I and P-Loop Mutations from Molecular Dynamics Simulations.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2917-2917
Author(s):  
Tai-Sung Lee ◽  
Steven Potts ◽  
Hagop Kantarjian ◽  
Jorge Cortes ◽  
Francis Giles ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Electrostatic Interactions ◽  
Large Scale ◽  
Resistance Mechanism ◽  
Md Simulations ◽  
Resistance Mechanisms ◽  
Static Structure ◽  
Imatinib Resistance ◽  
Dynamics Simulations

Abstract Molecular dynamics (MD) simulations on the complex of imatinib with the wild-type, T315I, and other 10 P-loop mutants of the tyrosine kinase Bcr-Abl have been performed to study the imatinib resistance mechanism at the atomic level. MD simulations show that large scale computational simulations could offer insight information that a static structure or simple homology modeling methods cannot provide for studying the Bcr-Abl imatinib resistance problem, especially in the case of conformational changes due to remote mutations. By utilizing the Molecular Mechanics/Poisson-Boltzmann surface area (MM-PBSA) techniques and analyzing the interactions between imatinib and individual residues, imatinib resistance mechanisms not previously thought have been revealed. Non-directly contacted P-loop mutations either unfavorably change the direct electrostatic interactions with imatinib, or cause the conformational changes influencing the contact energies between imatinib and other non-P-loop residues. We demonstrate that imatinib resistance of T315I mainly comes from the breakdown of the interactions between imatinib and E286 and M290, contradictory to previously suggested that the missing hydrogen bonding is the main contribution. We also demonstrate that except for the mutations of the direct contact residues, such as L248 and Y253, the unfavorable electrostatic interaction between P-loop and imatinib is the main reason for resistance for the P-loop mutations. Furthermore, in Y255H, protonation of the histidin is essential for rendering this mutation resistant to Gleevec. Our results demonstrate that MD is a powerful way to verify and predict clinical response or resistance to imatinib and other potential drugs.

scholarly journals Combining Virtual Reality Visualization with Ensemble Molecular Dynamics to Study Complex Protein Conformational Changes

2020 ◽  
Author(s):  
Jordi Juárez-Jiménez ◽  
Philip Tew ◽  
Michael o'connor ◽  
Salome Llabres ◽  
Rebecca Sage ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Virtual Reality ◽  
Conformational Changes ◽  
Large Scale ◽  
Computational Cost ◽  
Md Simulations ◽  
Collective Variables ◽  
Protein Conformational Changes ◽  
Sampling Protocols ◽  
State Models

<p>Molecular dynamics (MD) simulations are increasingly used to elucidate relationships between protein structure, dynamics and their biological function. Currently it is extremely challenging to perform MD simulations of large-scale structural rearrangements in proteins that occur on millisecond timescales or beyond, as this requires very significant computational resources, or the use of cumbersome ‘collective variable’ enhanced sampling protocols. Here we describe a framework that combines ensemble MD simulations and virtual-reality visualization (eMD-VR) to enable users to interactively generate realistic descriptions of large amplitude, millisecond timescale protein conformational changes in proteins. Detailed tests demonstrate that eMD-VR substantially decreases the computational cost of folding simulations of a WW domain, without the need to define collective variables <i>a priori</i>. We further show that eMD-VR generated pathways can be combined with Markov State Models to describe the thermodynamics and kinetics of large-scale loop motions in the enzyme cyclophilin A. Our results suggest eMD-VR is a powerful tool for exploring protein energy landscapes in bioengineering efforts. </p>

scholarly journals Molecular dynamics simulations disclose early stages of the photo-activation of cryptochrome 4

2018 ◽  
Author(s):  
D. R. Kattnig ◽  
C. Nielsen ◽  
I. A. Solov’yov
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Large Scale ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Quantum Processes ◽  
Helical Segment ◽  
High Betweenness ◽  
Dynamics Simulations ◽  
Fad Binding

AbstractBirds appear to be equipped with a light-dependent, radical-pair-based magnetic compass that relies on truly quantum processes. While the identity of the sensory protein has remained speculative, cryptochrome 4 has recently been identified as the most auspicious candidate. Here, we report on allatom molecular dynamics (MD) simulations addressing the structural reorganisations that accompany the photoreduction of the flavin cofactor in a model of the European robin cryptochrome 4 (ErCry4). Extensive MD simulations reveal that the photo-activation of ErCry4 induces large-scale conformational changes on short (hundreds of nanoseconds) timescales. Specifically, the photo-reduction is accompanied with the release of the C-terminal tail, structural rearrangements in the vicinity of the FAD-binding site, and the noteworthy formation of an α-helical segment at the N-terminal part. Some of these rearrangements appear to expose potential phosphorylation sites. We describe the conformational dynamics of the protein using a graph-based approach that is informed by the adjacency of residues and the correlation of their local motions. This approach reveals densely coupled reorganisation communities, which facilitate an efficient signal transduction due to a high density of hubs. These communities are interconnected by a small number of highly important residues characterized by high betweenness centrality. The network approach clearly identifies the sites restructuring upon photoactivation, which appear as protrusions or delicate bridges in the reorganisation network. We also find that, unlike in the homologous cryptochrome from D. melanogaster, the release of the C-terminal domain does not appear to be correlated with the transposition of a histidine residue close to the FAD cofactor.

scholarly journals Changes in active site histidine hydrogen bonding trigger cryptochrome activation

2016 ◽  
Vol 113 (36) ◽  
pp. 10073-10078 ◽  
Author(s):  
Abir Ganguly ◽  
Craig C. Manahan ◽  
Deniz Top ◽  
Estella F. Yee ◽  
Changfan Lin ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Conformational Changes ◽  
Large Scale ◽  
Ph Dependence ◽  
Md Simulations ◽  
Cellular Assays ◽  
Hydrogen Bonding Pattern ◽  
Poisson Boltzmann ◽  
Active Site Histidine ◽  
Clock Protein

Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.

scholarly journals Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

2020 ◽  
Vol 16 (11) ◽  
pp. e1008293
Author(s):  
Dylan Girodat ◽  
Avik K. Pati ◽  
Daniel S. Terry ◽  
Scott C. Blanchard ◽  
Karissa Y. Sanbonmatsu
Keyword(s):  
Time Scales ◽  
Single Molecule ◽  
Conformational Changes ◽  
Time Resolution ◽  
Large Scale ◽  
Md Simulations ◽  
Orientation Factor ◽  
Ribonucleoprotein Complexes ◽  
Fluorescence Measurements

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

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