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G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β(2)-adrenergic receptor (β(2)AR) and the cholecystokinin (CCK) receptor subfamily. The β(2)AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β(2)AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK(1)R shows functional sensitivity to membrane cholesterol while CCK(2)R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β(2)AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-T(m)) shows that mutation of a conserved CRAC sequence on TM7 of the β(2)AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid‐Polyamine‐organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo‐electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo‐EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all‐atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward‐facing conformation. Comparisons of the resulting Bor1p model with the X‐ray structure of AE1 in an outward‐facing conformation, together with MD simulations of inward‐facing and outward‐facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking‐bundle transport mechanism described for other members of the APC superfamily.

Summary Ligandbook is a public database and archive for force field parameters of small and drug-like molecules. It is a repository for parameter sets that are part of published work but are not easily available to the community otherwise. Parameter sets can be downloaded and immediately used in molecular dynamics simulations. The sets of parameters are versioned with full histories and carry unique identifiers to facilitate reproducible research. Text-based search on rich metadata and chemical substructure search allow precise identification of desired compounds or functional groups. Ligandbook enables the rapid set up of reproducible molecular dynamics simulations of ligands and protein-ligand complexes. Availability and Implementation Ligandbook is available online at https://ligandbook.org and supports all modern browsers. Parameters can be searched and downloaded without registration, including access through a programmatic RESTful API. Deposition of files requires free user registration. Ligandbook is implemented in the PHP Symfony2 framework with TCL scripts using the CACTVS toolkit. Supplementary information Supplementary data are available at Bioinformatics online.

Transmembrane proteins are responsible for transporting ions and small molecules across the hydrophobic region of the cell membrane. We are reviewing the evidence for regulation of these transport processes by interactions with the lipids of the membrane. We focus on ion channels, including potassium channels, mechanosensitive and pentameric ligand gated ion channels, and active transporters, including pumps, sodium or proton driven secondary transporters and ABC transporters. For ion channels it has been convincingly shown that specific lipid-protein interactions can directly affect their function. In some cases, a combined approach of molecular and structural biology together with computer simulations has revealed the molecular mechanisms. There are also many transporters whose activity depends on lipids but understanding of the molecular mechanisms is only beginning.

Escherichia coli NhaA is a prototype sodium-proton antiporter, which has been extensively characterized by X-ray crystallography, biochemical and biophysical experiments. However, the identities of proton carriers and details of pH-regulated mechanism remain controversial. Here we report constant pH molecular dynamics data, which reveal that NhaA activation involves a net charge switch of a pH sensor at the entrance of the cytoplasmic funnel and opening of a hydrophobic gate at the end of the funnel. The latter is triggered by charging of Asp164, the first proton carrier. The second proton carrier Lys300 forms a salt bridge with Asp163 in the inactive state, and releases a proton when a sodium ion binds Asp163. These data reconcile current models and illustrate the power of state-of-the-art molecular dynamics simulations in providing atomic details of proton-coupled transport across membrane which is challenging to elucidate by experimental techniques., The pH dependence of the activity of Escherichia coli main sodium-proton antiporter NhaA is still not fully understood. Here, the authors use continuous constant pH molecular dynamics simulations to identify NhaA proton carrier residues and elucidate its gating and ion transport processes.

Transient receptor potential (TRP) ion channels are eukaryotic polymodal sensors that function as molecular cellular signal integrators. TRP family members sense and are modulated by a wide array of inputs, including temperature, pressure, pH, voltage, chemicals, lipids, and other proteins. These inputs induce signal transduction events mediated by nonselective cation passage through TRP channels. In this review, we focus on the thermosensitive TRP channels and highlight the emerging view that these channels play a variety of significant roles in physiology and pathophysiology in addition to sensory biology. We attempt to use this viewpoint as a framework to understand the complexity and controversy of TRP channel modulation and ultimately suggest that the complex functional behavior arises inherently because this class of protein is exquisitely sensitive to many diverse and distinct signal inputs. To illustrate this idea, we primarily focus on TRP channel thermosensing. We also offer a structural, biochemical, biophysical, and computational perspective that may help to bring more coherence and consensus in understanding the function of this important class of proteins.

Ion channels are gated, i.e. they can switch conformation between a closed and an open state. Molecular dynamics simulations may be used to study the conformational dynamics of ion channels and of simple channel models. Simulations on model nanopores reveal that a narrow (

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

The evolutionary relationships of membrane transport proteins of the nucleobase-cation-symport (NCS-1) family from bacteria, fungi, and plants are described. The reported substrates of the NCS-1 family include nucleobases, hydantoins, and vitamins. Secondary active transport of substrate accompanied by a sodium ion, or possibly a proton, is the usual mechanism of energization. A strategy for the amplified expression, purification, and activity assays of bacterial members of the NCS-1 family is described. Conditions are given for the production of diffracting crystals of one member, the Na+-coupled transporter for aromatic hydantoins, ‘Mhp1’, from Microbacterium liquefaciens. The 3D structures of three forms of the Mhp1 protein are discussed in terms of one open-outward, one substrate-occluded, and one open-inward conformation contributing to a molecular dynamics simulation of the alternating-access model of membrane transport. The unexpected similarity of the protein fold of Mhp1 to those of transport proteins, hitherto thought to be from different evolutionary families, is discussed. 3D Structure Keywords: membrane transport protein; transporter structure; nucleobase; hydantoin

Volume 1 IRON Heme Proteins: Oxygen Storage and Oxygen Transport Proteins Heme Proteins: Cytochromes Heme Proteins: Cytochrome Peroxidases Heme Proteins: Cytochrome P-450 Heme Proteins: Oxidoreductases Non-Heme Proteins: Iron-Sulfur Clusters Non-Heme Proteins: Mononuclear Iron Proteins Volume 2 IRON continued Non-Heme Proteins: Dinuclear Iron Proteins Non-Heme Proteins: Iron Storage Non-Heme Proteins: Iron Transport NICKEL MANGANESE COBALT MOLYBDENUM/TUNGSTEN COPPER Cupredoxins (Type-1 Copper Proteins) Type-2 Copper Enzymes Binuclear Copper: Type-3 Copper Enzymes Binuclear Copper: CuA Copper Multicopper Enzymes Copper Storage and Transport VANADIUM.

Previous studies have reported that the KcsA potassium channel has an osmotic permeability coefficient of 4.8 × 10−12 cm3/s, giving it a significantly higher osmotic permeability coefficient than that of some membrane channels specialized in water transport. This high osmotic permeability is proposed to occur when the channel is depleted of potassium ions, the presence of which slow down the water permeation process. The atomic structure of the potassium-depleted KcsA channel and the mechanisms of water permeation have not been well characterized so far. Here, all-atom molecular dynamics simulations, in conjunction with an umbrella sampling strategy and a nonequilibrium approach to simulate pressure gradients are employed to illustrate the permeation of water in the absence of ions through the KcsA K+ channel. Equilibrium molecular dynamics simulations (95 ns combined total length) identified a possible structure of the potassium-depleted KcsA channel, and umbrella sampling calculations (160 ns combined total length) revealed that this structure is not permeable by water molecules moving along the channel axis. The simulation of a pressure gradient across the channel (30 ns combined total length) identified an alternative permeation pathway with a computed osmotic permeability of ∼(2.7 ± 0.9) × 10−13 cm3/s. Water fluxes along this pathway did not proceed through collective water motions or transitions to vapor state. All of the major results of this study were robust against variations in a wide set of simulation parameters (force field, water model, membrane model, and channel conformation). Proteins 2009. © 2008 Wiley-Liss, Inc.

Na þ /H þ antiporters are vital to cells for maintaining homeostasis, especially in high-salt environments. New crystal structures for two such antiporters, cytoplasmic-open Escherichia coli NhaA and periplasmic-open Thermus Thermophilus NapA, show high structural similarity despite low sequence identity. Among their common features are a set of highly conserved charged residues (three aspartates and one lysine) near the putative ion binding site. Using molecular dynamics simulations, we observe that interaction with sodium is dependent on the charge states of the conserved aspartates. The lysine and the nearest asparate also form a previously unidentified salt bridge. Under simulated physiological pH the presence of a sodium ion disrupts and breaks the salt bridge in NhaA. Given the presence of this salt bridge in both protein structures, its behavior under simulation, and the known importance of the conserved lysine, we hypothesize that this salt bridge is directly involved in ion binding and transport. To address the question of sodium binding, we performed an ensemble of all-atom equilibrium molecular dyanamics simulations (over 10 ms in total), varying the protonation states of the conserved residues and quantifying the resulting sodium interaction. To address the question of proton binding, we performed heuristic pKa calculations on this ensemble of simulations, which support the hypothesis that the lysine binds protons in a sodium-dependent manner. In order to elucidate the structural basis for alternating access in NhaA, we created an outward-facing model of NhaA, based on the periplasmic-open NapA structure, and simulated conformational transitions of NhaA from its cytoplasmic- to periplasmic-open state using dynamic importance sampling MD. Taken together, the combination of recent structural and dynamic simulation data suggests a new model of ion binding and transport for the CPA2 class of antiporters. 987-Plat

A hydrophobic pore of subnanometer dimensions can appear impermeable to an ion even though its radius is still much wider than that of the ion. Pores of molecular dimensions can be found, for instance, in carbon nanotubes, zeolites, or ion channel proteins. We quantify this barrier to ion permeation by calculating the potential of mean force from umbrella-sampled molecular dynamics simulations and compare them to continuum-electrostatic Poisson−Boltzmann calculations. The latter fail to describe the ion barrier because they do not account for the properties of water in the pore. The barrier originates from the energetic cost to desolvate the ion. Even in wide pores, which could accommodate an ion and its hydration shell, a barrier of several kT remains because the liquid water phase is not stable in the hydrophobic pore. Thus, the properties of the solvent play a crucial role in determining permeation properties of ions in confinement at the molecular scale.

The cystic fibrosis transmembrane conductance regulator (CFTR) is the only known member of the ATP binding cassette transporter superfamily that functions as an ion channel. Its architecture is analogous to that of ABC exporters such as p-glycoprotein, MsbA and Sav1866, which export their substrates using an alternating access mechanism driven by catalytic cycles coupled to ATP hydrolysis. The opening and closing of the CFTR channel is also driven by ATP catalytic cycles. We studied the state-dependence of the reactivity of three engineered cysteines located at positions 334, 337 and 338 in transmembrane helix 6 (TM6) of CFTR toward externally-applied, thiol-directed reagents, in order to investigate the conformational changes associated with channel gating. The engineered cysteine at position 334 was more reactive in the closed state, consistent with the findings of Zhang et al. (2005; J. Biol. Chem. 280: 41997-42003), while the engineered cysteines at positions 337 and 338 were more reactive in the open state. These findings are consistent with the predictions of homology models of the CFTR channel based on the crystal structures of bacterial homologues captured in inward- and outward-facing states of the proteins. Funded by the National Institute of Diabetes and Digestive and Kidney Diseases, the Cystic Fibrosis Foundation, and the American Lung Association.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated, anion-selective channel that is the product of the gene mutated in the inherited disease, cystic fibrosis. Chloride conductance via CFTR is a key component of the salt and water secretory system that maintains mucus hydration in the airway lumen. CFTR belongs to the large family of ABC transporter proteins, but it is the only member known to function as an ion channel. The channel is highly selective for anions over cations, but the structural basis for the observed charge selectivity is unknown. Individual point mutations generally do not seem to strongly affect charge selectivity, suggesting the possibility of a mechanism that is not highly dependent on local structural specialization such as that seen in the K-channel selectivity filter. We used a recent structural model of CFTR, based on the crystal structure of the Sav1866 ABC transporter and experimentally validated by cysteine scanning [1], to begin a computational investigation of the structural basis of the selectivity of the CFTR channel for anions over cations. Poisson-Boltzmann calculations suggest that an excess of basic residues in the transmembrane domain creates a large, anion-stabilizing region that includes the channel pore and extends towards the extracellular vestibule. The results indicate that electrostatic interactions affecting a large volume of the protein are a major contributor to anion over cation selectivity of the CFTR channel, similar to the situation observed in OprP [2].[1] C Alexander et al. Biochemistry 48 (2009) 10078-10088.[2] P Pongprayoon et al PNAS 106 (2009), 21614-21618.