No abstract

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is at an intermediate redox state of this process containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form of hVKOR, whereas disruption of the Cys51-Cys132 disulfide impairs warfarin binding and causes warfarin resistance. Relying on binding interactions identified by cysteine alkylation footprinting and mass spectrometry coupled with mutagenesis analysis, we are able to conduct structure simulations to reveal a closed warfarin-binding pocket stabilized by the Cys51-Cys132 linkage. Understanding the selective warfarin inhibition of a specific redox state of hVKOR should enable the rational design of drugs that exploit the redox chemistry and associated conformational changes in hVKOR.

The recently developed GROMOS 54A7 force field, a modification of the 53A6 force field, is validated by simulating the folding equilibrium of two β-peptides which show different dominant folds, i.e., a 314-helix and a hairpin, using three different force fields, i.e., GROMOS 45A3, 53A6, and 54A7. The 54A7 force field stabilizes both folds, and the agreement of the simulated NOE atom-atom distances with the experimental NMR data is slightly improved when using the 54A7 force field, while the agreement of the (3)J couplings with experimental results remains essentially unchanged when varying the force field. The 54A7 force field developed to improve the stability of α-helical structures in proteins can thus be safely used in simulations of β-peptides.

Serotonin receptors (5-HT3AR) directly regulate gut movement, and drugs that inhibit 5-HT3AR function are used to control emetic reflexes associated with gastrointestinal pathologies and cancer therapies. The 5-HT3AR function involves a finely tuned orchestration of three domain movements that include the ligand-binding domain, the pore domain, and the intracellular domain. Here, we present the structure from the full-length 5-HT3AR channel in the apo-state determined by single-particle cryo-electron microscopy at a nominal resolution of 4.3 Å. In this conformation, the ligand-binding domain adopts a conformation reminiscent of the unliganded state with the pore domain captured in a closed conformation. In comparison to the 5-HT3AR crystal structure, the full-length channel in the apo-conformation adopts a more expanded conformation of all the three domains with a characteristic twist that is implicated in gating.

Highlights d SMAPs bind at an intersubunit pocket defined by all three PP2A subunits d DT-061 (SMAP) binding results in selective stabilization of PP2A-B56a heterotrimers d Stabilization of B56a heterotrimers biases PP2A toward substrates such as c-Myc d Accumulation of methylated, B56a heterotrimers, is a potential clinical biomarker

This investigation has elucidated a mechanism for development of macrophage foam cells when macrophages are incubated with native low density lipoprotein (LDL). LDL is believed to be the main source of cholesterol that accumulates in monocyte-derived macrophages within atherosclerotic plaques, but native LDL has not previously been shown to cause substantial cholesterol accumulation when incubated with macrophages. We have found that activation of human monocyte-derived macrophages with phorbol 12-myristate 13-acetate ( Engorgement of macrophages with cholesterol is the defining pathological characteristic of atherosclerotic plaques, the cause of most heart attacks and strokes. Cholesterol accumulation in macrophages not only contributes to cholesterol retention within the vessel wall, but also alters macrophage biology. Cholesterol-loaded macrophages secrete plaque-disrupting matrix metalloproteinases, and produce tissue factor that promotes thrombosis when plaques rupture (1-3). Thus, how macrophages accumulate cholesterol and become foam cells has been the subject of intense investigation.Low density lipoprotein (LDL), 1 the main carrier of plasma cholesterol, enters the vessel wall and then by some mechanism enters macrophages. Previously, native LDL could not be shown to cause foam cell formation because the cellular receptor that binds LDL is poorly expressed on differentiated macrophages and down-regulates during cholesterol uptake, limiting total cholesterol accumulation (4 -7). Moreover, the LDL receptor is not expressed in human atherosclerotic plaques (8). Thus, most previous studies of macrophage foam cell formation have focused on modifying LDL in some way that increases its binding to macrophages. Increased macrophage binding of LDL has been achieved with chemical modifications to the apoB component of the LDL, aggregation of LDL induced by either vortexing or treatment of LDL with lipases, and complexing of LDL with other molecules, for example, glycosaminoglycans or antibodies, which bind macrophages and promote LDL uptake by piggyback endocytosis (9). Macrophages take up modified LDL by receptormediated endocytosis in pinocytotic vesicles, phagocytic vacuoles, or patocytic surface-connected compartments (9).One popular hypothesis of foam cell formation involves LDL oxidation. LDL oxidation promotes macrophage LDL uptake that is mediated by various macrophage scavenger receptors (10). Although oxidation of LDL has important biological effects that could influence atherosclerotic plaque development (11), oxidation of LDL does not readily explain foam cell formation. Incubation of human monocyte-derived macrophages with oxidized LDL, even strongly oxidized with artificial chemical systems, produces little macrophage cholesterol accumulation (12, 13). Also, oxidized LDL is poorly metabolized within lysosomes of macrophages because of partial inactivation by oxidized LDL of the lysosomal enzymes that degrade LDL (14 -16). This limits the capacity of oxidized LDL to induce acyl-CoA:cholesterol acyltra...

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

We have measured the dynamics of solvation of a triplet state probe, quinoxaline, in the glass-forming ionic liquid dibutylammonium formate near its glass transition temperature Tg=153 K. The Stokes-shift correlation function displays a relaxation time dispersion of considerable magnitude and the optical line width changes systematically along the solvation coordinate. The solvent dynamics in the viscous regime is compared with the rotational behavior of the solute and with the dielectric relaxation of the ionic liquid. Among the different quantities derived from the dielectric experiments, the relaxation of the macroscopic electric field, i.e., the modulus Mt, matches best the solvent response Ct regarding time scale and stretching exponent. Many other properties are reminiscent of the behavior of polar molecular liquids which lack the ionic character.