Insights into the molecular basis of action of the AT1 antagonist losartan using a combined NMR spectroscopy and computational approach.

The drug:membrane interactions for the antihypertensive AT1 antagonist losartan, the prototype of the sartans class, are studied herein using an integrated approach. The pharmacophore arrangement of the drug was revealed by rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) NMR spectroscopy in three different environments, namely water, dimethyl sulfoxide (DMSO), and sodium dodecyl sulfate (SDS) micellar solutions mimicking conditions of biological transport fluids and membrane lipid bilayers. Drug association with micelles was monitored by diffusion ordered spectroscopy (2D DOSY) and drug:micelle intermolecular interactions were characterized by ROESY spectroscopy. The localisation of the drug in the micellar environment was investigated by introducing 5-doxyl and 16-doxyl stearic acids. The use of spin labels confirmed that losartan resides close to the micelle:water interface with the hydroxymethyl group and the tetrazole heterocyclic aromatic ring facing the polar surface with the potential to interact with SDS charged polar head groups in order to increase amphiphilic interactions. The spontaneous insertion, the diffusion pathway and the conformational features of losartan were monitored by Molecular Dynamics (MD) simulations in a modeled SDS micellar aggregate environment and a long exploratory MD run (580ns) in a phospholipid dipalmitoylphosphatidylcholine (DPPC) bilayer with the AT1 receptor embedded. MD simulations were in excellent agreement with experimental results and further revealed the molecular basis of losartan:membrane interactions in atomic-level detail. This applied integrated approach aims to explore the role of membranes in losartan's pathway towards the AT1 receptor.

Ligand-protein docking studies of potential HIV-1 drug compounds using the algorithm FlexX.

Four compounds are docked to a pentameric bundle representing the transmembrane part of the Vpu protein from HIV-1. Employing the docking algorithm FlexX, their free energy of binding is estimated leading to the conclusion that potential drug candidates need to form H-bonds either with neighbouring or with n + 2 helices at the site of the serines within the bundle.

Model generation of viral channel forming 2B protein bundles from polio and coxsackie viruses.

2B is a 99 amino acid membrane protein encoded by enteroviruses such as polio and coxsackie viruses with two transmembrane domains. The protein is found to make membranes of infected cells permeable. Using a computational approach which positions the models and assesses stability by molecular dynamics (MD) simulations a putative tetrameric bundle model of 2B is generated. The bundles show a pore lining motif of three lysines followed by a serine. The bundle is discussed in terms of different possible orientations of the helices in the membrane and the consequences this has on the in vivo activity of 2B.

Reconstructing potentials of mean force from short steered molecular dynamics simulations of Vpu from HIV-1.

Vpu from human immunodeficiency virus type-1 (HIV-1) is an 81 amino acid type I integral membrane protein. Vpu forms ion conducting homooligomeric assemblies. To assess the energy landscape of an ion traversing the channel or pore single ion potentials of mean force (PMF) are reconstructed from short (1.2 ns) steered molecular dynamics (SMD) simulations using the Langevin equation of motion. For the simulations a section of the first 32 amino acids including the transmembrane domain of the Vpu protein is used. The values for the friction coefficient are estimated as a function of time using the velocity autocorrelation method. The PMFs of K(+), Na(+), and C(-) adopt a wave like pattern with a maximum around the hydrophobic stretch of the pore and a minimum at the hydrophilic site (C terminus). Independent of the pore size the amplitude of the PMF of at least one cation is always the lowest.

Protein-protein interactions: modeling the hepatitis C virus ion channel p7.

The p7 protein is a small ion-channel-forming membrane polypeptide encoded by the hepatitis C virus which consists of two transmembrane alpha-helices, TM1 and TM2, and can be blocked by long-alkyl-chain iminosugar derivatives. The length of TM1 and TM2 was estimated by employing different secondary structure prediction algorithms and is proposed to span from Ala-10 to Leu-32 for TM1 and from Trp-36 to Pro-58 for TM2. A configurational search protocol based on simulated annealing combined with short restrained molecular dynamics simulations is used in addition to protein-protein docking to investigate the packing of TM1/TM2. Full p7 oligomeric bundles were generated, and in the most plausible models serines and threonines are facing the hydrophilic pore. In these models, His-17 would be a pore-facing residue, suggesting that p7 may be sensitive to pH in respect to its function.

Molecular dynamics simulations of GlpF in a micelle vs in a bilayer: conformational dynamics of a membrane protein as a function of environment.

Octyl glucoside (OG) is a detergent widely employed in structural and functional studies of membrane proteins. To better understand the nature of protein-OG interactions, molecular dynamics simulations (duration 10 ns) have been used to explore an alpha-helical membrane protein, GlpF, in OG micelles and in DMPC bilayers. Greater conformational drift of the extramembraneous protein loops, from the initial X-ray structure, is seen for the GlpF-OG simulations than for the GlpF-DMPC simulation. The mobility of the transmembrane alpha-helices is approximately 1.3x higher in the GlpF-OG than the GlpF-DMPC simulations. The detergent is seen to form an irregular torus around the protein. The presence of the protein leads to a small perturbation in the behavior of the alkyl chains in the OG micelle, namely an approximately 15% increase in the trans-gauche(-)-gauche(+) transition time. Aromatic side chains (Trp, Tyr) and basic side chains (Arg, Lys) play an important role in both protein-detergent (OG) and protein-lipid (DMPC) interactions.

Membrane protein structure quality in molecular dynamics simulation.

Our goal was to assess the relationship between membrane protein quality, output from protein quality checkers and output from molecular dynamics (MD) simulations. Membrane transport proteins are essential for a wide range of cellular processes. Structural features of integral membrane proteins are still under-explored due to experimental limitations in structure determination. Computational techniques can be used to exploit biochemical and medium resolution structural data, as well as sequence homology to known structures, and enable us to explore the structure-function relationships in several transmembrane proteins. The quality of the models produced is vitally important to obtain reliable predictions. An examination of the relationship between model stability in molecular dynamics (MD) simulations derived from RMSD (root mean squared deviation) and structure quality assessment from various protein quality checkers was undertaken. The results were compared to membrane protein structures, solved at various resolution, by either X-ray or electron diffraction techniques. The checking programs could predict the potential success of MD in making functional conclusions. MD stability was shown to be a good indicator for the quality of structures. The quality was also shown to be dependent on the resolution at which the structures were determined.

Conformational sampling and dynamics of membrane proteins from 10-nanosecond computer simulations.

In the current report, we provide a quantitative analysis of the convergence of the sampling of conformational space accomplished in molecular dynamics simulations of membrane proteins of duration in the order of 10 nanoseconds. A set of proteins of diverse size and topology is considered, ranging from helical pores such as gramicidin and small beta-barrels such as OmpT, to larger and more complex structures such as rhodopsin and FepA. Principal component analysis of the C(alpha)-atom trajectories was employed to assess the convergence of the conformational sampling in both the transmembrane domains and the whole proteins, while the time-dependence of the average structure was analyzed to obtain single-domain information. The membrane-embedded regions, particularly those of small or structurally simple proteins, were found to achieve reasonable convergence. By contrast, extra-membranous domains lacking secondary structure are often markedly under-sampled, exhibiting a continuous structural drift. This drift results in a significant imprecision in the calculated B-factors, which detracts from any quantitative comparison to experimental data. In view of such limitations, we suggest that similar analyses may be valuable in simulation studies of membrane protein dynamics, in order to attach a level of confidence to any biologically relevant observations.

Water in ion channels and pores--simulation studies.

The microscopic properties of water in narrow pores are relevant to the function of ion channels and related membrane transport proteins. The emergence of several high-resolution structures allows one to perform molecular dynamics simulation studies of water in such pores. Simulations of bundles of parallel alpha-helical peptides (e.g. alamethicin) have enabled development of methodologies and concepts appropriate to such investigations. In the narrow channels formed by such bundles, water molecules exhibit reduced rotational and translation motion. This reduction in water mobility may be a general property of narrow pores. We have used simplified channel models to explore the role of hydrophobicity/hydrophilicity in the entry of water into pores. Narrow pores with a hydrophobic lining, although physically open, may not admit water molecules, acting as a 'hydrophobic gate' that prevents water and ion permeation. Such a gate can be opened either by widening the pore or making its lining more polar. Simulations have been used to explore the behaviour of water in GlpF, a member of the aquaporin family of water pores, and OmpA, a bacterial outer membrane protein. Preliminary results suggest that a continuous water wire is not formed within the amphipathic GlpF pore. Simulations of OmpA, in which polar residues line the channel, indicate that a small conformational change in one of the channel lining side chains may open the channel. In summary, comparison of the behaviour of water in different narrow transmembrane pores suggests that an amphipathic pore is ideal for water permeation, and that either a highly hydrophobic pore lining or a charged pore-lining region can act as a gate.

Structural characterization and computer-aided optimization of a small-molecule inhibitor of the Arp2/3 complex, a key regulator of the actin cytoskeleton.

CK-666 (1) is a recently discovered small-molecule inhibitor of the actin-related protein 2/3 (Arp2/3) complex, a key actin cytoskeleton regulator with roles in bacterial pathogenesis and cancer cell motility. Although 1 is commercially available, the crystal structure of Arp2/3 complex with 1 bound has not been reported, making its mechanism of action uncertain. Furthermore, its relatively low potency increases its potential for off-target effects in vivo, complicating interpretation of its influence in cell biological studies and precluding its clinical use. Herein we report the crystal structure of 1 bound to Arp2/3 complex, which reveals that 1 binds between the Arp2 and Arp3 subunits to stabilize the inactive conformation of the complex. Based on the crystal structure, we used computational docking and free-energy perturbation calculations of monosubstituted derivatives of 1 to guide optimization efforts. Biochemical assays of ten newly synthesized compounds led to the identification of compound 2, which exhibits a threefold increase in inhibitory activity in vitro relative to 1. In addition, our computational analyses unveiled a surface groove at the interface of the Arp2 and Arp3 subunits that can be exploited for additional structure-based optimization.