Conformational dynamics of adenylate kinase in crystals.

Adenylate kinase is a ubiquitous enzyme in living systems and undergoes dramatic conformational changes during its catalytic cycle. For these reasons, it is widely studied by genetic, biochemical, and biophysical methods, both experimental and theoretical. We have determined the basic crystal structures of three differently liganded states of adenylate kinase from Methanotorrus igneus, a hyperthermophilic organism whose adenylate kinase is a homotrimeric oligomer. The multiple copies of each protomer in the asymmetric unit of the crystal provide a unique opportunity to study the variation in the structure and were further analyzed using advanced crystallographic refinement methods and analysis tools to reveal conformational heterogeneity and, thus, implied dynamic behaviors in the catalytic cycle.

PCA-Plus: Enhanced principal component analysis with illustrative applications to batch effects and their quantitation.

Background: Principal component analysis (PCA), a standard approach to analysis and visualization of large datasets, is commonly used in biomedical research for detecting similarities and differences among groups of samples. We initially used conventional PCA as a tool for critical quality control of batch and trend effects in multi-omic profiling data produced by The Cancer Genome Atlas (TCGA) project of the NCI. We found, however, that conventional PCA visualizations were often hard to interpret when inter-batch differences were moderate in comparison with intra-batch differences; it was also difficult to quantify batch effects objectively. We, therefore, sought enhancements to make the method more informative in those and analogous settings. Results: We have developed algorithms and a toolbox of enhancements to conventional PCA that improve the detection, diagnosis, and quantitation of differences between or among groups, e.g., groups of molecularly profiled biological samples. The enhancements include (i) computed group centroids; (ii) sample-dispersion rays; (iii) differential coloring of centroids, rays, and sample data points; (iii) trend trajectories; and (iv) a novel separation index (DSC) for quantitation of differences among groups. Conclusions: PCA-Plus has been our most useful single tool for analyzing, visualizing, and quantitating batch effects, trend effects, and class differences in molecular profiling data of many types: mRNA expression, microRNA expression, DNA methylation, and DNA copy number. An early version of PCA-Plus has been used as the central graphical visualization in our MBatch package for near-real-time surveillance of data for analysis working groups in more than 70 TCGA, PanCancer Atlas, PanCancer Analysis of Whole Genomes, and Genome Data Analysis Network projects of the NCI. The algorithms and software are generic, hence applicable more generally to other types of multivariate data as well. PCA-Plus is freely available in a down-loadable R package at our MBatch website.

Exploring the impact of athletic identity on gender role conflict and athlete injury fear avoidance in male English professional academy football players.

Men's academy football can encourage a commitment to the athletic role and masculine norms. When injured, the ability to fulfil an athletic masculine identity is threatened and athletes may experience injury fear-avoidance behaviours as part of a negative injury appraisal. The aim of the study was to explore whether higher athletic identity (AI) was associated with higher gender role conflict and injury-related fear-avoidance. Seventy-two male English academy footballers completed an Athletic Identity Measurement Scale (AIMS), Gender Role Conflict Scale (GRCS), and Athlete Fear Avoidance Questionnaire (AFAQ) based on self-reported historical injuries. Correlational analyses were conducted for all variables, and a one-way ANOVA was used to compare high, moderate, and low AI. AIMS was significantly positively correlated with two GRCS subscales: success, power, and competition (SPC) and restricted affectionate behaviour between men (RAM). AIMS exclusivity also positively correlated with SPC and AIMS negative affectivity positively correlated with GRCS total and RAM. Additionally, the current study showed that high and moderate levels of AI had significantly higher levels of total GRCS than those with low AI. No significant results were found for AIMS, GRCS, and AFAQ. Results suggest that players with higher and more exclusive AI may be susceptible to masculine role conflicts, specifically, SPC and RAM, especially when there is a risk to their athletic role. The current study informs sport and health professionals of the need to monitor AI and masculine conformity in academy-level footballers to minimise gender-role conflict and potential maladaptive rehabilitation responses when their identities are threatened.

Evidence of Coevolution Between Cronartium harknessii Lineages and Their Corresponding Hosts, Lodgepole Pine and Jack Pine.

Variation in rate of infection and susceptibility of Pinus spp. to the fungus Cronartium harknessii (syn. Endocronartium harknessii), the causative agent of western gall rust, has been well documented. To test the hypothesis that there is a coevolutionary relationship between C. harknessii and its hosts, we examined genetic structure and virulence of C. harknessii associated with lodgepole pine (P. contorta var. latifolia), jack pine (P. banksiana), and their hybrids. A secondary objective was to improve assessment and diagnosis of infection in hosts. Using 18 microsatellites, we assessed genetic structure of C. harknessii from 90 sites within the ranges of lodgepole pine and jack pine. We identified two lineages (East and West, FST = 0.677) associated with host genetic structure (r = 0.81, P = 0.001), with East comprising three sublineages. In parallel, we conducted a factorial experiment in which lodgepole pine, jack pine, and hybrid seedlings were inoculated with spores from the two primary genetic lineages. With this experiment, we refined the phenotypic categories associated with infection and demonstrated that stem width can be used as a quantitative measure of host response to infection. Overall, each host responded differentially to the fungal lineages, with jack pine exhibiting more resiliency to infection than lodgepole pine and hybrids exhibiting intermediate resiliency. Taken together, the shared genetic structure between fungus and host species, and the differential interaction of the fungal species with the hosts, supports a coevolutionary relationship between host and pathogen.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Signatures of Post-Glacial Genetic Isolation and Human-Driven Migration in the Dothistroma Needle Blight Pathogen in Western Canada.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Persistent Protein Motions in a Rugged Energy Landscape Revealed by Normal Mode Ensemble Analysis.

Proteins are allosteric machines that couple motions at distinct, often distant, sites to control biological function. Low-frequency structural vibrations are a mechanism of this long-distance connection and are often used computationally to predict correlations, but experimentally identifying the vibrations associated with specific motions has proved challenging. Spectroscopy is an ideal tool to explore these excitations, but measurements have been largely unable to identify important frequency bands. The result is at odds with some previous calculations and raises the question what methods could successfully characterize protein structural vibrations. Here we show the lack of spectral structure arises in part from the variations in protein structure as the protein samples the energy landscape. However, by averaging over the energy landscape as sampled using an aggregate 18.5 µs of all-atom molecular dynamics simulation of hen egg white lysozyme and normal-mode analyses, we find vibrations with large overlap with functional displacements are surprisingly concentrated in narrow frequency bands. These bands are not apparent in either the ensemble averaged vibrational density of states or isotropic absorption. However, in the case of the ensemble averaged anisotropic absorption, there is persistent spectral structure and overlap between this structure and the functional displacement frequency bands. We systematically lay out heuristics for calculating the spectra robustly, including the need for statistical sampling of the protein and inclusion of adequate water in the spectral calculation. The results show the congested spectrum of these complex molecules obscures important frequency bands associated with function and reveal a method to overcome this congestion by combining structurally sensitive spectroscopy with robust normal mode ensemble analysis.

Lipids Alter Rhodopsin Function via Ligand-like and Solvent-like Interactions.

Rhodopsin, a prototypical G protein-coupled receptor, is a membrane protein that can sense dim light. This highly effective photoreceptor is known to be sensitive to the composition of its lipidic environment, but the molecular mechanisms underlying this fine-tuned modulation of the receptor's function and structural stability are not fully understood. There are two competing hypotheses to explain how this occurs: 1) lipid modulation occurs via solvent-like interactions, where lipid composition controls membrane properties like hydrophobic thickness, which in turn modulate the protein's conformational equilibrium; or 2) protein-lipid interactions are ligand-like, with specific hot spots and long-lived binding events. By analyzing an ensemble of all-atom molecular dynamics simulations of five different states of rhodopsin, we show that a local ordering effect takes place in the membrane upon receptor activation. Likewise, docosahexaenoic acid acyl tails and phosphatidylethanolamine headgroups behave like weak ligands, preferentially binding to the receptor in inactive-like conformations and inducing subtle but significant structural changes.

Selectivity and Mechanism of Fengycin, an Antimicrobial Lipopeptide, from Molecular Dynamics.

Fengycin is a cyclic lipopeptide used as an agricultural fungicide. It is synthesized by Bacillus subtilis as an immune response against fungal infection and functions by damaging the target's cell membrane. Previous molecular dynamics simulations and experiments have led to the hypothesis that the aggregation of fengycins on the membrane surface plays a key role in cell disruption. Here, we used microsecond-scale all-atom molecular dynamics simulations to understand the specificity, selectivity, and structure of fengycin oligomers. Our simulations suggest that fengycin is more likely to form stable oligomers in model fungal membranes (phosphatidylcholine) compared to the model bacterial membranes (phosphatidylethanolamine:phosphatidylglycerol). Furthermore, we characterize the differences in the structure and kinetics of the membrane-bound aggregates and discuss their functional implications.

A Galaxy Implementation of Next-Generation Clustered Heatmaps for Interactive Exploration of Molecular Profiling Data.

Clustered heatmaps are the most frequently used graphics for visualization of molecular profiling data in biology. However, they are generally rendered as static, or only modestly interactive, images. We have now used recent advances in web technologies to produce interactive "next-generation" clustered heatmaps (NG-CHM) that enable extreme zooming and navigation without loss of resolution. NG-CHMs also provide link-outs to additional information sources and include other features that facilitate deep exploration of the biology behind the image. Here, we describe an implementation of the NG-CHM system in the Galaxy bioinformatics platform. We illustrate the algorithm and available computational tool using RNA-seq data from The Cancer Genome Atlas program's Kidney Clear Cell Carcinoma project. Cancer Res; 77(21); e23-26. ©2017 AACR.

Minimal Nucleation State of α-Synuclein Is Stabilized by Dynamic Threonine-Water Networks.

The first structures of α-synuclein (αSyn) fibrils have recently been solved. Here, we use a unique combination of molecular dynamics simulation strategies to address the minimal nucleation size of the 11-amino acid NAC protofibril solved by X-ray and to interrogate the dynamic behavior of unexpected crystal waters in the steric zipper. We found that protofibrils of >8 chains are thermodynamically stabilized due to protection of the fibril core from solvent influx and ordering of the end strands by the fibril core. In these stable oligomers, water molecules resolved in the crystal structure freely exchange with bulk solvent but are, on average, stably coordinated along the ß-sheet by inward-facing Thr72 and Thr75. We confirm the persistence of this water coordination via simulations of the full-length Greek-key structure solved by NMR and speculate that these Thr-water networks are important in the context of enhanced fibril nucleation in the familial A53T mutation.

Retinal Conformation Changes Rhodopsin's Dynamic Ensemble.

G protein-coupled receptors are vital membrane proteins that allosterically transduce biomolecular signals across the cell membrane. However, the process by which ligand binding induces protein conformation changes is not well understood biophysically. Rhodopsin, the mammalian dim-light receptor, is a unique test case for understanding these processes because of its switch-like activity; the ligand, retinal, is bound throughout the activation cycle, switching from inverse agonist to agonist after absorbing a photon. By contrast, the ligand-free opsin is outside the activation cycle and may behave differently. We find that retinal influences rhodopsin dynamics using an ensemble of all-atom molecular dynamics simulations that in aggregate contain 100 µs of sampling. Active retinal destabilizes the inactive state of the receptor, whereas the active ensemble was more structurally homogenous. By contrast, simulations of an active-like receptor without retinal present were much more heterogeneous than those containing retinal. These results suggest allosteric processes are more complicated than a ligand inducing protein conformational changes or simply capturing a shifted ensemble as outlined in classic models of allostery.

Prolonged Exposure Dermatosis: Reporting High Incidence of an Undiagnosed Facial Dermatosis on a Winter Wilderness Expedition.

OBJECTIVE: Previously unclassified inflammatory skin lesions referred to as sun bumps have been observed throughout the year on participants of wilderness trips; however, the underlying cause and diagnosis remain unclear. The purpose of this prospective observational study was to document the incidence, characteristics, and risk factors associated with these skin lesions as they occurred on a winter wilderness expedition. METHODS: For this study, the lesions were defined as pruritic or erythematous skin lesions occurring while in the wilderness. Seventy-four participants in a wilderness ski touring course in Wyoming fully completed a 44-question written survey concerning occurrence and risk factors for these lesions. Weather information and photographs were collected. RESULTS: Twenty-six percent of participants had similar lesions. The lesions were described as edematous pale papules and plaques with erosions and crusts on an erythematous background. The face was involved in 90% of affected persons. Lesions occurred after an average of 8.7 days in the wilderness and resolved 10.6 days later. Skin that was less prone to sunburn was associated with a decreased incidence (odds ratio 0.44). No association could be found between lesion incidence and history of polymorphous light eruption, sun exposure, ambient temperature, affected contacts, sex, or body mass index. CONCLUSIONS: Overall, the lesions were common among study participants but occurred only after prolonged exposure to wilderness conditions. It was not possible to classify the skin condition as an example of any known diagnosis. We propose the name "prolonged exposure dermatosis" for this condition until further studies better define its etiology, prevention, and treatment.

Relationships among muscle dysmorphia characteristics, body image quality of life, and coping in males.

OBJECTIVES: The purpose of this study was to examine relationships among bodybuilding dependence, muscle satisfaction, body image-related quality of life and body image-related coping strategies, and test the hypothesis that muscle dysmorphia characteristics may predict quality of life via coping strategies. DESIGN: Participants (294 males, Mage=20.5 years, SD=3.1) participated in a cross-sectional survey. METHODS: Participants completed questionnaires assessing muscle satisfaction, bodybuilding dependence, body image-related quality of life and body image-related coping. RESULTS: Quality of life was correlated positively with muscle satisfaction and bodybuilding dependence but negatively with body image coping (P<0.05). Body image coping was correlated positively with bodybuilding dependence and negatively with muscle satisfaction (P<0.05). Mediation analysis found that bodybuilding dependence and muscle satisfaction predicted quality of life both directly and indirectly via body image coping strategies (as evidenced by the bias corrected and accelerated bootstrapped confidence intervals). CONCLUSIONS: These results provide preliminary evidence regarding the ways that muscularity concerns might influence body image-related quality of life.

Lightweight object oriented structure analysis: tools for building tools to analyze molecular dynamics simulations.

LOOS (Lightweight Object Oriented Structure-analysis) is a C++ library designed to facilitate making novel tools for analyzing molecular dynamics simulations by abstracting out the repetitive tasks, allowing developers to focus on the scientifically relevant part of the problem. LOOS supports input using the native file formats of most common biomolecular simulation packages, including CHARMM, NAMD, Amber, Tinker, and Gromacs. A dynamic atom selection language based on the C expression syntax is included and is easily accessible to the tool-writer. In addition, LOOS is bundled with over 140 prebuilt tools, including suites of tools for analyzing simulation convergence, three-dimensional histograms, and elastic network models. Through modern C++ design, LOOS is both simple to develop with (requiring knowledge of only four core classes and a few utility functions) and is easily extensible. A python interface to the core classes is also provided, further facilitating tool development.

Structure-based simulations reveal concerted dynamics of GPCR activation.

G protein-coupled receptors (GPCRs) are a vital class of proteins that transduce biological signals across the cell membrane. However, their allosteric activation mechanism is not fully understood; crystal structures of active and inactive receptors have been reported, but the functional pathway between these two states remains elusive. Here, we use structure-based (Go-like) models to simulate activation of two GPCRs, rhodopsin and the ß2 adrenergic receptor (ß2AR). We used data-derived reaction coordinates that capture the activation mechanism for both proteins, showing that activation proceeds through quantitatively different paths in the two systems. Both reaction coordinates are determined from the dominant concerted motions in the simulations so the technique is broadly applicable. There were two surprising results. First, the main structural changes in the simulations were distributed throughout the transmembrane bundle, and not localized to the obvious areas of interest, such as the intracellular portion of Helix 6. Second, the activation (and deactivation) paths were distinctly nonmonotonic, populating states that were not simply interpolations between the inactive and active structures. These transitions also suggest a functional explanation for ß2AR's basal activity: it can proceed through a more broadly defined path during the observed transitions.

Retinal ligand mobility explains internal hydration and reconciles active rhodopsin structures.

Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state (2)H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp265(6.48) residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

Simulating the mechanism of antimicrobial lipopeptides with all-atom molecular dynamics.

The emergence of antibiotic resistant pathogens is one of the major medical concerns of the 21st century, prompting renewed interest in the development of novel antimicrobial compounds. Here we use microsecond-scale all-atom molecular dynamics simulations to characterize the structure, dynamics, and membrane-binding mechanism of a synthetic antimicrobial lipopeptide, C16-KGGK. Our simulations suggest that these lipopeptides prefer to aggregate in solution and alter the intrinsic order of the lipid bilayer upon binding. From these results and previous coarse-grained simulations, we have developed a simple model for the binding and insertion process for these lipopeptides.