Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

Nicotinic acetylcholine receptors are prototypes for the pharmaceutically important family of pentameric ligand-gated ion channels. Here we present atomic resolution structures of nicotine and carbamylcholine binding to AChBP, a water-soluble homolog of the ligand binding domain of nicotinic receptors and their family members, GABAA, GABAC, 5HT3 serotonin, and glycine receptors. Ligand binding is driven by enthalpy and is accompanied by conformational changes in the ligand binding site. Residues in the binding site contract around the ligand, with the largest movement in the C loop. As expected, the binding is characterized by substantial aromatic and hydrophobic contributions, but additionally there are close contacts between protein oxygens and positively charged groups in the ligands. The higher affinity of nicotine is due to a main chain hydrogen bond with the B loop and a closer packing of the aromatic groups. These structures will be useful tools for the development of new drugs involving nicotinic acetylcholine receptor-associated diseases.

The 2.1-Å resolution crystal structure of wild-type green f luorescent protein and comparison of it with the recently determined structure of the Ser-65 3 Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the f luorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the f luorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the f luorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the f luorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the f luorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green f luorescent protein should enable engineering of environmentally sensitive f luorescent indicators and UV-triggered f luorescent markers of protein diffusion and trafficking in living cells.The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is the first known protein in which visible fluorescence is genetically encodable. The fluorophore is derived from natural residues present within the primary structure of GFP, so no exogenous cofactor or substrate is needed for fluorescence (1, 2). The tremendous potential of GFP as a reporter of gene expression, cell lineage, and protein trafficking and interactions has been extensively reviewed (3-5).Wild-type (WT) GFP is a 238-aa protein (2). In vitro GFP is a particularly stable protease-resistant protein (6) and is only denatured under extreme conditions (7). The GFP chromophore, p-hydroxybenzylideneimidazolinone (8, 9), is formed by internal cyclization of a Ser-Tyr-Gly tripeptide and 1,2-dehydrogenation of the Tyr. This posttranslational modification is oxygendependent, requiring Ϸ2-4 h for the WT protein (10, 11). A mechanism for the fluorophore formation has been proposed (3) but needs to be confirmed by further studies.GFP absorbs blue light at 395 nm, with a smaller peak at 475 nm, and emits green light at 508 nm with a quantum yie...

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5′-triphosphate (ATP) turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.