Hv1 voltage-gated proton channels mediate rapid and selective transmembrane H+ flux and are gated by both voltage and pH gradients. Selective H+ transfer in membrane proteins is commonly achieved by Grotthuss proton ‘hopping’ in chains of ionizable amino acid side chains and intraprotein water molecules. To identify whether ionizable residues are required for proton permeation in Hv1, we neutralized candidate residues and measured expressed voltage-gated H+ currents. Unexpectedly, charge neutralization was insufficient to abrogate either the Hv1 conductance or coupling of pH gradient and voltage-dependent activation. Molecular dynamics simulations revealed water molecules in the central crevice of Hv1 model structures but not in homologous voltage-sensor domain (VSD) structures. Our results indicate that Hv1 most likely forms an internal water wire for selective proton transfer and that interactions between water molecules and S4 arginines may underlie coupling between voltage- and pH-gradient sensing.

An X-ray crystal structure of Kelch-like ECH-associated protein (Keap1) co-crystallised with (1S,2R)-2-[(1S)-1-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,2,3,4-tetrahydroisoquinolin-2-carbonyl]cyclohexane-1-carboxylic acid (compound (S,R,S)-1 a) was obtained. This X-ray crystal structure provides breakthrough experimental evidence for the true binding mode of the hit compound (S,R,S)-1 a, as the ligand orientation was found to differ from that of the initial docking model, which was available at the start of the project. Crystallographic elucidation of this binding mode helped to focus and drive the drug design process more effectively and efficiently.

The synaptic vesicle glycoprotein SV2A belongs to the major facilitator superfamily (MFS) of transporters and is an integral constituent of synaptic vesicle membranes. SV2A has been demonstrated to be involved in vesicle trafficking and exocytosis, processes crucial for neurotransmission. The anti-seizure drug levetiracetam was the first ligand to target SV2A and displays a broad spectrum of anti-seizure activity in various preclinical models. Several lines of preclinical and clinical evidence, including genetics and protein expression changes, support an important role of SV2A in epilepsy pathophysiology. While the functional consequences of SV2A ligand binding are not fully elucidated, studies suggest that subsequent SV2A conformational changes may contribute to seizure protection. Conversely, the recently discovered negative SV2A modulators, such as UCB0255, counteract the anti-seizure effect of levetiracetam and display procognitive properties in preclinical models. More broadly, dysfunction of SV2A may also be involved in Alzheimer’s disease and other types of cognitive impairment, suggesting potential novel therapies for levetiracetam and its congeners. Furthermore, emerging data indicate that there may be important roles for two other SV2 isoforms (SV2B and SV2C) in the pathogenesis of epilepsy, as well as other neurodegenerative diseases. Utilization of recently developed SV2A positron emission tomography ligands will strengthen and reinforce the pharmacological evidence that SV2A is a druggable target, and will provide a better understanding of its role in epilepsy and other neurological diseases, aiding in further defining the full therapeutic potential of SV2A modulation.

Advances in nanomanipulation techniques have made it possible to measure the response of an individual biomolecule to a force applied in the laboratory. Experiments that stretch a single molecule of duplex DNA have been difficult to interpret theoretically, particularly as the major changes in molecular structure caused by the force cannot be measured. In principle, computer simulation can calculate these conformational changes in atomic level detail, but to date such studies have failed to reproduce the experimental data due to the computational expense of the calculations. Here we show that a combination of molecular modeling and statistical physics can be used successfully to understand the stretching behavior of DNA. Our simulations provide new information about the dynamics of DNA denaturation under force in atomic level detail and also show the importance of entropy in determining biomechanical properties in general.

A novel series of methyl ester-terminated C8-linked pyrrolobenzodiazepine (PBD)-poly(N-methylpyrrole) conjugates (50a-f) has been synthesized and their DNA interaction evaluated by thermal denaturation, DNA footprinting, and in vitro transcription stop assays. The synergistic effect of attaching a PBD unit to a polypyrrole fragment is illustrated by the large increase in DNA binding affinity (up to 50-fold) compared to the individual PBD and pyrrole components. 50a-f were found to bind mainly to identical DNA sequences but with apparent binding site widths increasing with molecular length and the majority of sites conforming to the consensus motif 5'-XGXWz (z = 3 +/- 1; W = A or T; X = any base but preferably a purine). They also provided robust sequence-selective blockade of transcription at sites corresponding approximately to their DNA footprints. 50a-f were shown to have good cellular/nuclear penetration properties, and a degree of correlation between cytotoxicity and DNA-binding affinity was observed.

The adenosine A 2A receptor (A 2A R) has long been implicated in cardiovascular disorders. As more selective A 2A R ligands are being identified, its roles in other disorders, such as Parkinson's disease, are starting to emerge, and A 2A R antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A 2A receptor bound to compound 1 (Cmpd-1), a novel A 2A R/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A 2A receptor with a cytochrome b562-RIL (BRIL) fusion (A 2A R-BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A 2A R-BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1-bound A 2A R-BRIL prevented formation of the lattice observed with the ZM241385-bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A 2A R structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr9 1.35 and Tyr271 7.36 , which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A 2A R structures, highlighting flexibility in the binding pocket that may facilitate the development of A 2A R-selective compounds for the treatment of Parkinson's disease.T he adenosine A 2A receptor (A 2A R) is one of the four subtypes of adenosine receptors (A 1 R, A 2A R, A 2B R, A 3 R). As a member of the G protein-coupled receptor (GPCR) family, the A 2A receptor couples to stimulatory G protein G s and elevates intracellular cAMP upon activation by endogenous adenosine. The A 2A R has been an attractive drug target due to its role in cardiovascular and immune system function, as well as in the central nervous system as a potential therapeutic target for Parkinson's disease (PD) (1-4). PD is a neurodegenerative disease that affects more than 1% of the population over 65 years old. Currently, major treatments target the restoration of dopamine signaling, which is impaired in PD patients, by dopamine-replacing agents. Although these treatments effectively address PD-related motor disturbances, the long-term use of dopamine-replacing agents is associated with the development of motor complications; therefore, there is a need for nondopaminergic drugs (2). It is known that A 2A R signaling regulates dopaminergic neurotransmission, and A 2A R antagonists have been shown to enhance D2 dopamine receptor signaling, which has been found to improve PD symptoms in animal models and patients, without the side effects common to dopaminereplacing agents, such...

SummaryThe nature of voltage sensing by voltage-activated ion channels is a key problem in membrane protein structural biology. The way in which the voltage-sensor (VS) domain interacts with its membrane environment remains unclear. In particular, the known structures of Kv channels do not readily explain how a positively charged S4 helix is able to stably span a lipid bilayer. Extended (2 × 50 ns) molecular dynamics simulations of the high-resolution structure of the isolated VS domain from the archaebacterial potassium channel KvAP, embedded in zwitterionic and in anionic lipid bilayers, have been used to explore VS/lipid interactions at atomic resolution. The simulations reveal penetration of water into the center of the VS and bilayer. Furthermore, there is significant local deformation of the lipid bilayer by interactions between lipid phosphate groups and arginine side chains of S4. As a consequence of this, the electrostatic field is “focused” across the center of the bilayer.

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.