An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore

Genetic Variability and the Classification of Hepatitis E Virus (vol 87, pg 4161, 2013)

The classification of hepatitis E virus (HEV) variants is currently in transition without agreed definitions for genotypes and sub-types or for deeper taxonomic groupings into species and genera that could incorporate more recently characterized viruses as-signed to theHepeviridae family that infect birds, bats, rodents, and fish. These conflicts arise because of differences in the vi-ruses and genomic regions compared and in the methodology used. We have reexamined published sequences and found that synonymous substitutions were saturated in comparisons between and within virus genotypes. Analysis of complete genome sequences or concatenated ORF1/ORF2 amino acid sequences indicated that HEV variants most closely related to those infecting humans can be consistently divided into six genotypes (types 1 to 4 and two additional genotypes fromwild boar). Variants iso-lated from rabbits, closely related to genotype 3, occupy an intermediate position. No consistent criteria could be defined for the assignment of virus subtypes. Analysis of amino acid sequences from these viruses with the more divergent variants from chick

Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Percussion Convocation

Program listing performers and works performe

Consensus proposals for classification of the family hepeviridae

The family Hepeviridae consists of positive-stranded RNA viruses that infect a wide range of mammalian species, as well as chickens and trout. A subset of these viruses infects humans and can cause a self-limiting acute hepatitis that may become chronic in immunosuppressed individuals. Current published descriptions of the taxonomical divisions within the family Hepeviridae are contradictory in relation to the assignment of species and genotypes. Through analysis of existing sequence information, we propose a taxonomic scheme in which the family is divided into the genera Orthohepevirus (all mammalian and avian hepatitis E virus (HEV) isolates) and Piscihepevirus (cutthroat trout virus). Species within the genus Orthohepevirus are designated Orthohepevirus A (isolates from human, pig, wild boar, deer, mongoose, rabbit and camel), Orthohepevirus B (isolates from chicken), Orthohepevirus C (isolates from rat, greater bandicoot, Asian musk shrew, ferret and mink) and Orthohepevirus D (isolates from bat). Proposals are also made for the designation of genotypes within the human and rat HEVs. This hierarchical system is congruent with hepevirus phylogeny, and the three classification levels (genus, species and genotype) are consistent with, and reflect discontinuities in the ranges of pairwise distances between amino acid sequences. Adoption of this system would include the avoidance of host names in taxonomic identifiers and provide a logical framework for the assignment of novel variants

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

The Hepatitis E Virus Polyproline Region Is Involved in Viral Adaptation

Genomes of hepatitis E virus (HEV), rubivirus and cutthroat virus (CTV) contain a region of high proline density and low amino acid (aa) complexity, named the polyproline region (PPR). In HEV genotypes 1, 3 and 4, it is the only region within the non-structural open reading frame (ORF1) with positive selection (4–10 codons with dN/dS>1). This region has the highest density of sites with homoplasy values >0.5. Genotypes 3 and 4 show ∼3-fold increase in homoplastic density (HD) in the PPR compared to any other region in ORF1, genotype 1 does not exhibit significant HD (p<0.0001). PPR sequence divergence was found to be 2-fold greater for HEV genotypes 3 and 4 than for genotype 1. The data suggest the PPR plays an important role in host-range adaptation. Although the PPR appears to be hypervariable and homoplastic, it retains as much phylogenetic signal as any other similar sized region in the ORF1, indicating that convergent evolution operates within the major HEV phylogenetic lineages. Analyses of sequence-based secondary structure and the tertiary structure identify PPR as an intrinsically disordered region (IDR), implicating its role in regulation of replication. The identified propensity for the disorder-to-order state transitions indicates the PPR is involved in protein-protein interactions. Furthermore, the PPR of all four HEV genotypes contains seven putative linear binding motifs for ligands involved in the regulation of a wide number of cellular signaling processes. Structure-based analysis of possible molecular functions of these motifs showed the PPR is prone to bind a wide variety of ligands. Collectively, these data suggest a role for the PPR in HEV adaptation. Particularly as an IDR, the PPR likely contributes to fine tuning of viral replication through protein-protein interactions and should be considered as a target for development of novel anti-viral drugs

Plutonic foundation of a slow-spreading ridge segment : oceanic core complex at Kane Megamullion, 23°30′N, 45°20′W

Author Posting. © American Geophysical Union, 2008. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geochemistry Geophysics Geosystems 9 (2008): Q05014, doi:10.1029/2007GC001645.We mapped the Kane megamullion, an oceanic core complex on the west flank of the Mid-Atlantic Ridge exposing the plutonic foundation of a ∼50 km long, second-order ridge segment. The complex was exhumed by long-lived slip on a normal-sense detachment fault at the base of the rift valley wall from ∼3.3 to 2.1 Ma (Williams, 2007). Mantle peridotites, gabbros, and diabase dikes are exposed in the detachment footwall and in outward facing high-angle normal fault scarps and slide-scar headwalls that cut through the detachment. These rocks directly constrain crustal architecture and the pattern of melt flow from the mantle to and within the lower crust. In addition, the volcanic carapace that originally overlay the complex is preserved intact on the conjugate African plate, so the complete internal and external architecture of the paleoridge segment can be studied. Seafloor spreading during formation of the core complex was highly asymmetric, and crustal accretion occurred largely in the footwall of the detachment fault exposing the core complex. Because additions to the footwall, both magmatic and amagmatic, are nonconservative, oceanic detachment faults are plutonic growth faults. A local volcano and fissure eruptions partially cover the northwestern quarter of the complex. This volcanism is associated with outward facing normal faults and possible, intersecting transform-parallel faults that formed during exhumation of the megamullion, suggesting the volcanics erupted off-axis. We find a zone of late-stage vertical melt transport through the mantle to the crust in the southern part of the segment marked by a ∼10 km wide zone of dunites that likely fed a large gabbro and troctolite intrusion intercalated with dikes. This zone correlates with the midpoint of a lineated axial volcanic high of the same age on the conjugate African plate. In the central region of the segment, however, primitive gabbro is rare, massive depleted peridotite tectonites abundant, and dunites nearly absent, which indicate that little melt crossed the crust-mantle boundary there. Greenschist facies diabase and pillow basalt hanging wall debris are scattered over the detachment surface. The diabase indicates lateral melt transport in dikes that fed the volcanic carapace away from the magmatic centers. At the northern edge of the complex (southern wall of the Kane transform) is a second magmatic center marked by olivine gabbro and minor troctolite intruded into mantle peridotite tectonite. This center varied substantially in size with time, consistent with waxing and waning volcanism near the transform as is also inferred from volcanic abyssal-hill relief on the conjugate African plate. Our results indicate that melt flow from the mantle focuses to local magmatic centers and creates plutonic complexes within the ridge segment whose position varies in space and time rather than fixed at a single central point. Distal to and between these complexes there may not be continuous gabbroic crust, but only a thin carapace of pillow lavas overlying dike complexes laterally fed from the magmatic centers. This is consistent with plate-driven flow that engenders local, stochastically distributed transient instabilities at depth in the partially molten mantle that fed the magmatic centers. Fixed boundaries, such as large-offset fracture zones, or relatively short segment lengths, however, may help to focus episodes of repeated melt extraction in the same location. While no previous model for ocean crust is like that inferred here, our observations do not invalidate them but rather extend the known diversity of ridge architecture.NSF Grants OCE-0118445, OCE-0624408 and OCE-0621660 supported this research. B. Tucholke was also supported by the Henry Bryant Bigelow Chair in Oceanography at Woods Hole Oceanographic Institution

Evolutionary History and Population Dynamics of Hepatitis E Virus

BACKGROUND: Hepatitis E virus (HEV) is an enterically transmitted hepatropic virus. It segregates as four genotypes. All genotypes infect humans while only genotypes 3 and 4 also infect several animal species. It has been suggested that hepatitis E is zoonotic, but no study has analyzed the evolutionary history of HEV. We present here an analysis of the evolutionary history of HEV. METHODS AND FINDINGS: The times to the most recent common ancestors for all four genotypes of HEV were calculated using BEAST to conduct a Bayesian analysis of HEV. The population dynamics for genotypes 1, 3 and 4 were analyzed using skyline plots. Bayesian analysis showed that the most recent common ancestor for modern HEV existed between 536 and 1344 years ago. The progenitor of HEV appears to have given rise to anthropotropic and enzootic forms of HEV, which evolved into genotypes 1 and 2 and genotypes 3 and 4, respectively. Population dynamics suggest that genotypes 1, 3 and 4 experienced a population expansion during the 20(th) century. Genotype 1 has increased in infected population size ∼30-35 years ago. Genotype 3 and 4 have experienced an increase in population size starting late in the 19(th) century until ca.1940-45, with genotype 3 having undergone additional rapid expansion until ca.1960. The effective population size for both genotype 3 and 4 rapidly declined to pre-expansion levels starting in ca.1990. Genotype 4 was further examined as Chinese and Japanese sequences, which exhibited different population dynamics, suggesting that this genotype experienced different evolutionary history in these two countries. CONCLUSIONS: HEV appears to have evolved through a series of steps, in which the ancestors of HEV may have adapted to a succession of animal hosts leading to humans. Analysis of the population dynamics of HEV suggests a substantial temporal variation in the rate of transmission among HEV genotypes in different geographic regions late in the 20(th) Century