Ion Binding and Internal Hydration in the Multidrug Resistance Secondary Active Transporter NorM Investigated by Molecular Dynamics Simulations

Recently, a 3.65 Å resolution structure of the transporter NorM from the multidrug and toxic compound extrusion family has been determined in the outward-facing conformation. This antiporter uses electrochemical gradients to drive substrate export of a large class of antibiotic and toxic compounds in exchange for small monovalent cations (H⁺ and Na⁺), but the molecular details of this mechanism are still largely unknown. Here we report all-atom molecular dynamics simulations of NorM, with and without the bound Na⁺ cation and at different ion concentrations. Spontaneous binding of Na⁺ is observed in several independent simulations with transient ion binding to D36 being necessary to reach the final binding site for which two competitive binding modes occur. Finally, the simulations indicate that the extracellular vestibule of the transporter invariably loses its characteristic V shape indicated by the crystallographic data, and it is reduced to a narrow permeation pathway lined by polar residues that can act as a specific pore for the transport of small cations. This event, together with the available structures of evolutionarily related transporters of the major facilitator superfamily (MFS), suggests that differences in the hydrophobic content of the extracellular vestibule may be characteristic of multidrug resistance transporters in contrast to substrate-selective members of the MFS

Rhodopsin Absorption from First Principles: Bypassing Common Pitfalls

Bovine rhodopsin is the most extensively studied retinal protein and is considered the prototype of this important class of photosensitive biosystems involved in the process of vision. Many theoretical investigations have attempted to elucidate the role of the protein matrix in modulating the absorption of retinal chromophore in rhodopsin, but, while generally agreeing in predicting the correct location of the absorption maximum, they often reached contradicting conclusions on how the environment tunes the spectrum. To address this controversial issue, we combine here a thorough structural and dynamical characterization of rhodopsin with a careful validation of its excited-state properties via the use of a wide range of state-of-the-art quantum chemical approaches including various flavors of time-dependent density functional theory (TDDFT), different multireference perturbative schemes (CASPT2 and NEVPT2), and quantum Monte Carlo (QMC) methods. Through extensive quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations, we obtain a comprehensive structural description of the chromophore–protein system and sample a wide range of thermally accessible configurations. We show that, in order to obtain reliable excitation properties, it is crucial to employ a sufficient number of representative configurations of the system. In fact, the common use of a single, ad hoc structure can easily lead to an incorrect model and an agreement with experimental absorption spectra due to cancelation of errors. Finally, we show that, to properly account for polarization effects on the chromophore and to quench the large blue-shift induced by the counterion on the excitation energies, it is necessary to adopt an enhanced description of the protein environment as given by a large quantum region including as many as 250 atoms

Generalized QM/MM Force Matching Approach Applied to the 11-cis Protonated Schiff Base Chromophore of Rhodopsin

We extended a previously developed force matching approach to systems with covalent QM/MM boundaries and describe its user-friendly implementation in the publicly available software package CPMD. We applied this approach to the challenging case of the retinal protonated Schiff base in dark state bovine rhodopsin. We were able to develop a highly accurate force field that is able to capture subtle structural changes within the chromophore that have a pronounced influence on the optical properties. The optical absorption spectrum calculated from configurations extracted from a MD trajectory using the new force field is in excellent agreement with QM/MM and experimental references

Free energy differences (in kcal/mol) for the described transformations.

<p>Error estimates are included.</p><p>^a Calculated as </p><p></p><p></p><p><mi>Δ</mi><mi>Δ</mi></p><p><mi>G</mi></p><p>MOP<mo>→</mo>HMP</p><p>exp<mo>.</mo></p><p></p><mo>=</mo><mi>Δ</mi><p><mi>G</mi></p><p>bind</p><p>HMP<mo stretchy="false">(</mo>exp<mo>.</mo><mo stretchy="false">)</mo></p><p></p><mo>−</mo><mi>Δ</mi><p><mi>G</mi></p><p>bind</p><p>MOP<mo stretchy="false">(</mo>exp<mo>.</mo><mo stretchy="false">)</mo></p><p></p><mo>=</mo><mi>R</mi><mi>T</mi>ln<p><mo>[</mo></p><p></p><p></p><p></p><p><mi>K</mi><mi>i</mi></p><p>HMP<mo stretchy="false">(</mo>exp<mo>.</mo><mo stretchy="false">)</mo></p><p></p><p></p><mo>/</mo><p></p><p><mi>K</mi><mi>i</mi></p><p>MOP<mo stretchy="false">(</mo>exp<mo>.</mo><mo stretchy="false">)</mo></p><p></p><p></p><p></p><p></p><mo>]</mo><p></p><mo>.</mo><p></p><p></p><p></p><p></p><p>Free energy differences (in kcal/mol) for the described transformations.</p

Molecular structures of morphine and hydromorphone.

<p>Molecular structures of morphine and hydromorphone.</p

Representative structure of the MD simulations for (A) the MOP-μOR and (B) the HMP-μOR complexes obtained from clustering analysis.

<p>The ligand carbon atoms are in orange. H-bonds and salt-bridges are shown in green and magenta dashed lines, respectively. For clarity hydrogen atoms of the ligands and the μOR residues are not shown. H297 is monoprotonated at the Nε atom.</p

Conformational change of μOR EL3 observed in the case of MOP binding.

<p>E310 in EL3 forms a salt-bridge with K233 (magenta dashed lines), which remains until the end of the simulation.</p

Arrangements of aromatic residues at the μOR orthosteric binding site upon binding with (A) MOP and with (B) HMP.

<p>Arrangements of aromatic residues at the μOR orthosteric binding site upon binding with (A) MOP and with (B) HMP.</p

Minimum distances between both morphinan drugs (MOP and HMP) and selected protein residues.

<p>Distances are given in Å and averaged over the finite temperature MD trajectories.</p><p>Minimum distances between both morphinan drugs (MOP and HMP) and selected protein residues.</p

Schematic model of the agonist-induced μOR conformational change into an active-like state.

<p>Schematic model of the agonist-induced μOR conformational change into an active-like state.</p