Impact of Lipid Composition and Receptor Conformation on the Spatio-temporal Organization of µ-Opioid Receptors in a Multi-component Plasma Membrane Model.

The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the µ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR.

The effect of a widespread cancer-causing mutation on the inactive to active dynamics of the B-Raf kinase.

Protein kinases play a key role in regulating cellular processes. Kinase dysfunction can lead to disease, making them an attractive target for drug design. The B-Raf kinase is a key target for the treatment of melanoma since a single mutation (V600E) is found in more than 50% of all malignant melanomas. Despite the importance of B-Raf in melanoma treatment, the molecular mechanism by which the mutation increases kinase activity remains elusive. Since kinases are tightly regulated by a conformational transition between an active and inactive state, which is difficult to capture experimentally, large-scale enhanced-sampling simulations are performed to examine the mechanism by which the V600E mutation enhances the activity of the B-Raf monomer. The results reveal that the mutation has a twofold effect. First, the mutation increases the barrier of the active to inactive transition trapping B-Raf in the active state. The mutation also increases the flexibility of the activation loop which might speed-up the rate-limiting step of phosphorylation. Both effects can be explained by the formation of salt-bridges with the Glu600 residue.

Multiple state transition interface sampling of alanine dipeptide in explicit solvent.

We have applied the recently developed multiple state transition interface sampling approach to alanine dipeptide in explicit water. We extract the rate constant matrix for configurational changes between each pair of metastable states. The results are comparable with values from previous literature and show that the method is applicable to biomolecular systems.

The effect of platinum on diffusion kinetics in beta-NiAl: implications for thermal barrier coating lifetimes.

Platinum is added to thermal barrier coatings (TBCs) as it is observed empirically to extend their lifetime, but the mechanism by which Pt acts is unknown. Since Pt has been proposed to alter diffusivities in NiAl, a key component of TBCs, we use first-principles quantum mechanics calculations to investigate atomic level diffusion mechanisms. Here, we examine the effect of Pt on five previously proposed mechanisms for Ni diffusion in NiAl: next-nearest-neighbor jumps, the triple defect mechanism, and three variants of the six jump cycle. We predict that Pt increases the rate of Ni diffusion by stabilizing point defects and defect clusters that are diffusion intermediates. Previously, we predicted the triple defect mechanism to be a dominant Ni diffusion mechanism; it simultaneously results in long-range Al diffusion in the opposite direction. Since Pt increases the rate of Ni diffusion, it also increases Al diffusion in NiAl, which may be key to extending the coating lifetime.

Investigating Small-Molecule Ligand Binding to G Protein-Coupled Receptors with Biased or Unbiased Molecular Dynamics Simulations.

An increasing number of G protein-coupled receptor (GPCR) crystal structures provide important-albeit static-pictures of how small molecules or peptides interact with their receptors. These high-resolution structures represent a tremendous opportunity to apply molecular dynamics (MD) simulations to capture atomic-level dynamical information that is not easy to obtain experimentally. Understanding ligand binding and unbinding processes, as well as the related responses of the receptor, is crucial to the design of better drugs targeting GPCRs. Here, we discuss possible ways to study the dynamics involved in the binding of small molecules to GPCRs, using long timescale MD simulations or metadynamics-based approaches.

Insights into the function of opioid receptors from molecular dynamics simulations of available crystal structures.

The opioid receptors are key targets in the treatment of acute and chronic pain, and the development of novel analgesics with reduced side effects is crucial in the search for more effective medications. The crystal structures of opioid receptors have provided a wealth of knowledge on many aspects of opioid receptor pharmacology and function, including ligand binding poses, location of the sodium allosteric binding site, conformational changes associated with activation and putative dimeric interfaces. These crystal structures also offer a starting point for molecular dynamics (MD) simulations to capture one aspect of drug design that static structures cannot resolve, namely protein dynamics. With the increase in computing power, MD simulations of crystal structures have become an influential tool in understanding the function of GPCRs in general. Here, we discuss lessons learned from MD simulations of opioid receptor crystal structures with reference to (i) the binding pathway of sodium to its crystallographic allosteric site, (ii) the dynamics of ligand-receptor and receptor-receptor interactions, both at the ligand- and G protein-binding sites, (iii) the binding pathway and binding pose of novel ligands, and (iv) opioid receptor oligomerization. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Folding dynamics of the Trp-cage miniprotein: evidence for a native-like intermediate from combined time-resolved vibrational spectroscopy and molecular dynamics simulations.

Trp-cage is a synthetic 20-residue miniprotein which folds rapidly and spontaneously to a well-defined globular structure more typical of larger proteins. Due to its small size and fast folding, it is an ideal model system for experimental and theoretical investigations of protein folding mechanisms. However, Trp-cage's exact folding mechanism is still a matter of debate. Here we investigate Trp-cage's relaxation dynamics in the amide I' spectral region (1530-1700 cm(-1)) using time-resolved infrared spectroscopy. Residue-specific information was obtained by incorporating an isotopic label ((13)C═(18)O) into the amide carbonyl group of residue Gly11, thereby spectrally isolating an individual 310-helical residue. The folding-unfolding equilibrium is perturbed using a nanosecond temperature-jump (T-jump), and the subsequent re-equilibration is probed by observing the time-dependent vibrational response in the amide I' region. We observe bimodal relaxation kinetics with time constants of 100 ± 10 and 770 ± 40 ns at 322 K, suggesting that the folding involves an intermediate state, the character of which can be determined from the time- and frequency-resolved data. We find that the relaxation dynamics close to the melting temperature involve fast fluctuations in the polyproline II region, whereas the slower process can be attributed to conformational rearrangements due to the global (un)folding transition of the protein. Combined analysis of our T-jump data and molecular dynamics simulations indicates that the formation of a well-defined α-helix precedes the rapid formation of the hydrophobic cage structure, implying a native-like folding intermediate, that mainly differs from the folded conformation in the orientation of the C-terminal polyproline II helix relative to the N-terminal part of the backbone. We find that the main free-energy barrier is positioned between the folding intermediate and the unfolded state ensemble, and that it involves the formation of the α-helix, the 310-helix, and the Asp9-Arg16 salt bridge. Our results suggest that at low temperature (T ≪ Tm) a folding path via formation of α-helical contacts followed by hydrophobic clustering becomes more important.

Confinement-induced states in the folding landscape of the Trp-cage miniprotein.

Although protein folding is typically studied in dilute solution, folding in a cell will be affected by interactions with other biomolecules and excluded volume effects. Here, we examine the effect of hydrophobic confinement on folding of the Trp-cage miniprotein. We used replica exchange molecular dynamics simulations to probe the differences between folding in the bulk, on a hydrophobic surface, and confined between two hydrophobic walls. In addition to promotion of helix formation due to reduced conformational entropy of the unfolded state upon confinement, adsorption of Trp-cage to a hydrophobic surface stabilizes intermediate structures not present in the bulk. These new intermediate structures may alter the folding mechanism and kinetics and show the importance of including environmental effects when studying protein folding.