DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a β-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.
Diastolic fluid dynamics in the left ventricle (LV) has been examined in multiple clinical studies for understanding cardiac function in healthy humans and developing diagnostic measures in disease conditions. The question of how intraventricular filling vortex flow pattern is affected by increasing heart rate (HR) is still unanswered. Previous studies on healthy subjects have shown a correlation between increasing HR and diminished E/A ratio of transmitral peak velocities during early filling (E-wave) to atrial systole (A-wave). We hypothesize that with increasing HR under constant E/A ratio, E-wave contribution to intraventricular vortex propagation is diminished. A physiologic in vitro flow phantom consisting of a LV physical model was used for this study. HR was varied across 70, 100 and 120 beats per minute (bpm) with E/A of 1.1-1.2. Intraventricular flow patterns were characterized using 2D particle image velocimetry measured across three parallel longitudinal (apical-basal) planes in the LV. A pair of counter-rotating vortices was observed during E-wave across all HRs. With increasing HR, diminished vortex propagation occurred during E-wave and atrial systole was found to amplify secondary vorticity production. The diastolic time point where peak vortex circulation occurred was delayed with increasing HR, with peak circulation for 120 bpm occurring as late as 90% into diastole near the end of A-wave. The role of atrial systole is elevated for higher HR due to the limited time available for filling. Our baseline findings and analysis approach can be applied to studies of clinical conditions where impaired exercise tolerance is observed.
Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.