The central cavity of <scp>ABCB</scp>1 undergoes alternating access during <scp>ATP</scp> hydrolysis

Wonderen, Jessica H. van; McMahon, Róisín M.; O’Mara, Megan L.; McDevitt, Christopher A.; Thomson, Andrew J.; Kerr, Ian D.; MacMillan, Fraser; Callaghan, Richard

doi:10.1111/febs.12773

Cited by 36 publications

(28 citation statements)

References 51 publications

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“…tariquidar, verapamil, doxorubicin, morphine) [20,36,41]. Furthermore, residues proximal to F978 (A980), and within the central cavity (F343), have been shown by EPR-spectroscopy to undergo a shift in accessibility to a polar environment as P-gp switches between conformations in the transport process [42]. Another recent investigation has…”

Section: Discussionmentioning

confidence: 99%

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

Mittra

Pavy

Subramanian

et al. 2017

Biochemical Pharmacology

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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.

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Section: Discussionmentioning

confidence: 99%

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

Mittra

Pavy

Subramanian

et al. 2017

Biochemical Pharmacology

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“…A single point mutation of cysteine covalently bound to the MTSL probe was incorporated into each of the PsaA systems, at residues L56, S58, I125, I236 and S266, as shown in Figure 1. The MTSL spin label was parameterized using the Automated Topology Builder (ATB) as described previously [33].…”

Section: Simulations Of Wild-type and Spin-labelled Psaamentioning

confidence: 99%

“…To de-couple the motion of the spin label from the conformational changes of the protein, the rotational angle of the probe relative to the Cα of the covalently bound cysteine was calculated. For this, the vector between the MTSL pyrrole nitrogen and the Cα of the covalently bound cysteine was calculated as a function of simulation time and the rotational angle of the probe was then calculated as the arccosine of the vector dot product, as described previously [33]. For each of the five MTSL-labelled PsaA variants, the time-dependent data of the rotational angle was binned into histograms and plotted as radial plots.…”

Section: Root Mean Squared Deviation (Rmsd)mentioning

confidence: 99%

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

Deplazes

Begg

Wonderen

et al. 2015

Biophysical Chemistry

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After the embargo period via non-commercial hosting platforms such as their institutional repository  via commercial sites with which Elsevier has an agreement In all cases accepted manuscripts should: link to the formal publication via its DOI  bear a CC-BY-NC-ND license -this is easy to do, click here to find out how  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy  not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article 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 the metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomicresolution 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. 4 GRAPHICAL ABSTRACT HIGHLIGHTS• PsaA is the Mn 2+ -recruiting protein of the human pathogen Streptococcus pneumoniae• Metal-free PsA samples structurally distinct conformations not captured by the crystal structure• The metal binding site is larger and more solvent exposed than indicated by the crystal structure KEYWORDSCluster A-I solute-binding protein (SBP); Streptococcus pneumoniae; PsaA; molecular dynamics simulations, electron paramagnetic resonance (EPR) spectroscopy; protein flexibility; spin-label mobility 5

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“…Correctly defining the path of conformational changes at the atomic level remains the biggest challenge. A recent investigation into the alternating access mechanism in ABCB1 has been performed using SDSL with cw-EPR spectroscopic methods and by rationalising this data with MD simulations to describe the conformational changes that the central cavity undergoes during translocation [11]. This investigation was driven by the recent structural data from the murine and Caenorhabditis elegans homologues of ABCB1.…”

Section: Abcb1mentioning

confidence: 99%

“…The EPR label has been parameterized for the most commonly used force fields CHARMM [54], Amber [55], GROMOS [11,56] and OPLS [57]. Literature reports indicate that the computational burden is very high for correct and complete sampling [58].…”

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confidence: 99%

Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

Stockner

Mullen

MacMillan

2015

Biochemical Society Transactions

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Abstract:ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented.

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The central cavity of ABCB1 undergoes alternating access during ATP hydrolysis

Cited by 36 publications

References 51 publications

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

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

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

Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications

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