Viral Packaging ATPases Utilize a Glutamate Switch to Couple ATPase Activity and DNA Translocation [preprint]

Many viruses utilize ringed packaging ATPases to translocate double-stranded DNA into procapsids during replication. A critical step in the mechanochemical cycle of such ATPases is ATP binding, which causes a subunit within the motor to grip DNA tightly. Here, we probe the underlying molecular mechanism by which ATP binding is coupled to DNA gripping and show that a glutamate switch residue found in AAA+ enzymes is central to this coupling in viral packaging ATPases. Using free energy landscapes computed through molecular dynamics simulations, we determined the stable conformational state of the ATPase active site in apo, ATP-bound, and ADP-bound states. Our results show that the catalytic glutamate residue transitions from an inactive to an active pose upon ATP binding, and that a residue assigned as the glutamate switch is necessary for regulating the transition. Further, we identified via mutual information analyses the intramolecular signaling pathway mediated by the glutamate switch that is responsible for coupling ATP binding to conformational transitions of DNA-gripping motifs. We corroborated these predictions with both structural and functional experimental data. Specifically, we showed that the crystal structure of the ADP-bound P74-26 packaging ATPase is consistent with the predicted structural coupling from simulations, and we further showed that disrupting the predicted signaling pathway indeed decouples ATPase activity from DNA translocation activity in the φ29 DNA packaging motor. Our work thus establishes a signaling pathway in viral DNA packaging motors that ensures coordination between chemical and mechanical events involved in viral DNA packaging

Atomistic Mechanism of Force Generation, Translocation, and Coordination in a Viral Genome Packaging Motor [preprint]

Double-stranded DNA viruses package their genomes into pre-assembled protein capsids using virally-encoded ATPase ring motors. While several structures of isolated monomers (subunits) from these motors have been determined, they provide little insight into how subunits within a functional ring coordinate their activities to efficiently generate force and translocate DNA. Here we describe the first atomic-resolution structure of a functional ring form of a viral DNA packaging motor and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Crystal structures of the pentameric ATPase ring from bacteriophage asccφ28 show that each subunit consists of a canonical N-terminal ASCE ATPase domain connected to a ‘vestigial’ nuclease domain by a small lid subdomain. The lid subdomain closes over the ATPase active site and engages in extensive interactions with a neighboring subunit such that several important catalytic residues are positioned to function in trans. The pore of the ring is lined with several positively charged residues that can interact with DNA. Simulations of the ATPase ring in various nucleotide-bound states provide information about how the motor coordinates sequential nucleotide binding, hydrolysis, and exchange around the ring. Simulations also predict that the ring adopts a helical structure to track DNA, consistent with recent cryo-EM reconstruction of the φ29 packaging ATPase. Based on these results, an atomistic model of viral DNA packaging is proposed wherein DNA translocation is powered by stepwise helical-to-planar ring transitions that are tightly coordinated by ATP binding, hydrolysis, and release

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccphi28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous phi29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases

Allosteric Antagonist Modulation of TRPV2 by Piperlongumine Impairs Glioblastoma Progression.

The use of computational tools to identify biological targets of natural products with anticancer properties and unknown modes of action is gaining momentum. We employed self-organizing maps to deconvolute the phenotypic effects of piperlongumine (PL) and establish a link to modulation of the human transient receptor potential vanilloid 2 (hTRPV2) channel. The structure of the PL-bound full-length rat TRPV2 channel was determined by cryo-EM. PL binds to a transient allosteric pocket responsible for a new mode of anticancer activity against glioblastoma (GBM) in which hTRPV2 is overexpressed. Calcium imaging experiments revealed the importance of Arg539 and Thr522 residues on the antagonistic effect of PL and calcium influx modulation of the TRPV2 channel. Downregulation of hTRPV2 reduces sensitivity to PL and decreases ROS production. Analysis of GBM patient samples associates hTRPV2 overexpression with tumor grade, disease progression, and poor prognosis. Extensive tumor abrogation and long term survival was achieved in two murine models of orthotopic GBM by formulating PL in an implantable scaffold/hydrogel for sustained local therapy. Furthermore, in primary tumor samples derived from GBM patients, we observed a selective reduction of malignant cells in response to PL ex vivo. Our results establish a broadly applicable strategy, leveraging data-motivated research hypotheses for the discovery of novel means tackling cancer

Structural Insights into the Quinolone Resistance Mechanism of Mycobacterium tuberculosis DNA Gyrase

Mycobacterium tuberculosis DNA gyrase, an indispensable nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and is hence the sole target for quinolone action, a crucial drug active against multidrug-resistant tuberculosis. To understand at an atomic level the quinolone resistance mechanism, which emerges in extensively drug resistant tuberculosis, we performed combined functional, biophysical and structural studies of the two individual domains constituting the catalytic DNA gyrase reaction core, namely the Toprim and the breakage-reunion domains. This allowed us to produce a model of the catalytic reaction core in complex with DNA and a quinolone molecule, identifying original mechanistic properties of quinolone binding and clarifying the relationships between amino acid mutations and resistance phenotype of M. tuberculosis DNA gyrase. These results are compatible with our previous studies on quinolone resistance. Interestingly, the structure of the entire breakage-reunion domain revealed a new interaction, in which the Quinolone-Binding Pocket (QBP) is blocked by the N-terminal helix of a symmetry-related molecule. This interaction provides useful starting points for designing peptide based inhibitors that target DNA gyrase to prevent its binding to DNA

Consistency of impact assessment protocols for non-native species

Standardized tools are needed to identify and prioritize the most harmful non-native species (NNS). A plethora of assessment protocols have been developed to evaluate the current and potential impacts of non-native species, but consistency among them has received limited attention. To estimate the consistency across impact assessment protocols, 89 specialists in biological invasions used 11 protocols to screen 57 NNS (2614 assessments). We tested if the consistency in the impact scoring across assessors, quantified as the coefficient of variation (CV), was dependent on the characteristics of the protocol, the taxonomic group and the expertise of the assessor. Mean CV across assessors was 40%, with a maximum of 223%. CV was lower for protocols with a low number of score levels, which demanded high levels of expertise, and when the assessors had greater expertise on the assessed species. The similarity among protocols with respect to the final scores was higher when the protocols considered the same impact types. We conclude that all protocols led to considerable inconsistency among assessors. In order to improve consistency, we highlight the importance of selecting assessors with high expertise, providing clear guidelines and adequate training but also deriving final decisions collaboratively by consensus

An update of the Worldwide Integrated Assessment (WIA) on systemic insecticides. Part 2: impacts on organisms and ecosystems

New information on the lethal and sublethal effects of neonicotinoids and fipronil on organisms is presented in this review, complementing the previous WIA in 2015. The high toxicity of these systemic insecticides to invertebrates has been confirmed and expanded to include more species and compounds. Most of the recent research has focused on bees and the sublethal and ecological impacts these insecticides have on pollinators. Toxic effects on other invertebrate taxa also covered predatory and parasitoid natural enemies and aquatic arthropods. Little, while not much new information has been gathered on soil organisms. The impact on marine coastal ecosystems is still largely uncharted. The chronic lethality of neonicotinoids to insects and crustaceans, and the strengthened evidence that these chemicals also impair the immune system and reproduction, highlights the dangers of this particular insecticidal classneonicotinoids and fipronil. , withContinued large scale – mostly prophylactic – use of these persistent organochlorine pesticides has the potential to greatly decreasecompletely eliminate populations of arthropods in both terrestrial and aquatic environments. Sublethal effects on fish, reptiles, frogs, birds and mammals are also reported, showing a better understanding of the mechanisms of toxicity of these insecticides in vertebrates, and their deleterious impacts on growth, reproduction and neurobehaviour of most of the species tested. This review concludes with a summary of impacts on the ecosystem services and functioning, particularly on pollination, soil biota and aquatic invertebrate communities, thus reinforcing the previous WIA conclusions (van der Sluijs et al. 2015)

Cervical lymph node metastasis in adenoid cystic carcinoma of the larynx: a collective international review

Adenoid cystic carcinoma (AdCC) of the head and neck is a well-recognized pathologic entity that rarely occurs in the larynx. Although the 5-year locoregional control rates are high, distant metastasis has a tendency to appear more than 5 years post treatment. Because AdCC of the larynx is uncommon, it is difficult to standardize a treatment protocol. One of the controversial points is the decision whether or not to perform an elective neck dissection on these patients. Because there is contradictory information about this issue, we have critically reviewed the literature from 1912 to 2015 on all reported cases of AdCC of the larynx in order to clarify this issue. During the most recent period of our review (1991-2015) with a more exact diagnosis of the tumor histology, 142 cases were observed of AdCC of the larynx, of which 91 patients had data pertaining to lymph node status. Eleven of the 91 patients (12.1%) had nodal metastasis and, based on this low proportion of patients, routine elective neck dissection is therefore not recommended

ARIA-EAACI statement on asthma and COVID-19 (June 2, 2020)

Non peer reviewe