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

Abstract

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.

Efficient Building Blocks for Solid‐Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

“…By comparing the spectra of all amino acids before (black) and after (blue) introduction of the spin label a broadening of all signals is obtained. This broadening is related to the paramagnetic character of the spin label and clearly indicates that the spin label is active . Furthermore, the comparison shows that all signals are preserved during the coupling step.…”

“…This broadening is related to the paramagnetic character of the spin label and clearly indicates that the spin label is active. [46][47][48][49][50] Furthermore, the comparison shows that all signals are preserved during the coupling step. Some signals are shifted which implies a proximity of these nuclei to the radical in the sample.…”

ABC transporter research: going strong 40 years on

“…Visualizing the structure of ABC transporters at atomic resolution has become something of a holy grail in the field but an understanding of their dynamics is equally important [ 46 ]. Ironically, the first ABC-related structure of a soluble SBP was published 10 years before the ABC transporter family was defined [ 47 ].…”

“…An armoury of complementary approaches is now available to probe ABC transporter structure and mechanism and these can be particularly powerful when combined. For example, van Veen discussed the use of cysteine-reactive fluorescent reagents to investigate the catalytic cycle of MsbA in the framework of crystal structures [ 55 ] and Fraser Macmillan [ 46 , 56 ] used P-gp to explain how EPR spectroscopy can be employed in combination with site-directed spin labelling to measure distances and probe accessibility in ABC transporters. Where structures are available, MD simulations, together with homology modelling, offer the possibility to describe conformational dynamics, albeit over short time scales.…”

“…Where structures are available, MD simulations, together with homology modelling, offer the possibility to describe conformational dynamics, albeit over short time scales. Laura Domicevica and Philip Biggin [ 57 ] presented an analysis of P-gp drug binding using homology modelling with MD and Thomas Stockner [ 46 ] discuss here how EPR and MD can be combined to investigate the conformational flexibility of ABC transporters, comparing dynamic data for MsbA and P-gp with mechanistic predictions from crystal structures in different conformations. A promising complementary technique was introduced by Bavro [ 58 ]; X-ray radiolytic footprinting combined MS (XF–MS) though not yet applied to ABC transporters, offers valuable insights into the dynamics of membrane proteins; in particular XF–MS permits identification of structural waters and conformational changes in proteins and is applicable to protein complexes, which will interest many members of the ABC transport community.…”