The molecular basis for sugar import in malaria parasites.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.

Exploring acoustic characteristics of different aircraft types by fusing with aircraft tracking data.

In this study, we develop a method that assigns acoustic signals with Automatic Dependent Surveillance-Broadcast (ADS-B) data to build a labeled dataset of acoustic signals from aircraft without expensive ground-truth experiments. An exploration of the resultant labeled dataset enables an assessment of the acoustic characteristics from three types of aircraft. The fusion framework is evaluated using data from an acoustic sensor and collocated ADS-B receiver in the middle of a large urban area at Southern Methodist University in Dallas, Texas. Our results demonstrate the benefit of combining multiple types of data to generate a labeled dataset leveraging open-source aircraft surveillance data. By studying three classes of aircraft, we find that the smaller fixed wing single engine (FWSE) class is mostly detected within approximately 5000 m, while the larger fixed wing multi-engine (FWME) class is commonly detected out to greater distances above 7500 m. The FWSE class has a median source frequency at 100 Hz, compared to FWME class with median source frequency at 80 Hz, while rotorcraft has a source frequency falling into a lower range of 30-100 Hz.

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing asparagine.

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation, and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMs structure and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

Spatial-resolution impacts on local infrasound propagation.

The accuracy of input meteorological data can significantly impact the successful prediction of infrasound propagation at local to near-regional distances. These meteorological inputs are often derived from weather model simulations when event-specific measurements are not available, but the ideal spatial resolutions of these simulations have not been determined. This study seeks to identify the ideal horizontal resolutions for input meteorological data via infrasound simulations conducted with both range-dependent and -independent inputs. Outputs from the Weather Research and Forecasting (WRF) model at 1, 3, 5, and 15 km horizontal resolutions enable these investigations. The parabolic equation propagation model is used to calculate transmission loss for an impulsive signal and is compared against experimental data obtained from a series of 1 lb spherical, suspended C4 shots recorded on the infrasound array on the Waterways Experiment Station in Vicksburg, MS, occurring throughout the diurnal cycle with an overall propagation distance of 14.5 km. Simulations for morning and nighttime correctly predict signal detection and non-detection. Transitional times of day (dawn, evening) were partially successful. Changing horizontal resolutions in WRF incurred greater differences in prediction results than use of range-dependence vs -independence. No clear picture emerged regarding the optimal horizontal resolution for meteorological inputs.

Numerical modeling of mesoscale infrasound propagation in the Arctic.

The impacts of characteristic weather events and seasonal patterns on infrasound propagation in the Arctic region are simulated numerically. The methodology utilizes wide-angle parabolic equation methods for a windy atmosphere with inputs provided by radiosonde observations and a high-resolution reanalysis of Arctic weather. The calculations involve horizontal distances up to 200 km for which interactions with the troposphere and lower stratosphere dominate. Among the events examined are two sudden stratospheric warmings, which are found to weaken upward refraction by temperature gradients while creating strongly asymmetric refraction from disturbances to the circumpolar winds. Also examined are polar low events, which are found to enhance negative temperature gradients in the troposphere and thus lead to strong upward refraction. Smaller-scale and topographically driven phenomena, such as low-level jets, katabatic winds, and surface-based temperature inversions, are found to create frequent surface-based ducting out to 100 km. The simulations suggest that horizontal variations in the atmospheric profiles, in response to changing topography and surface property transitions, such as ice boundaries, play an important role in the propagation.

Structural infrasound from a barge collision with the Mississippi river bridge.

The Mississippi River Bridge in Vicksburg, Mississippi, is a seven-span cantilever bridge that is 1033 m long by 20.9 m wide and is part of the Interstate-20 corridor. On March 23, 2011, at approximately 14:30 CST, a barge moving downstream struck a pier of the bridge. An infrasound array located at the U.S. Army Engineer Research and Development Center (ERDC) detected the impact. Finite Element (FE) models were developed to investigate the structural behavior of the bridge due to the impact. The FE models identified events within the infrasound record that were possibly produced by the different modes of vibration of the bridge structure. The emerging technology of structural infrasound monitoring and the potential for using infrasound as a forensic tool is demonstrated with the results of the bridge-barge collision event and capability of deployment in the future.

Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity.

Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactions with membrane proteins. Target receptors include several subtypes of pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolution. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulation. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motion and modulation upon modification of a transmembrane binding site within each GLIC subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation evident up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibition. These results support a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.

Infrasound monitoring in non-traditional environments.

To date, the infrasound community has avoided deployments in noisy urban sites because interests have been in monitoring distant sources with low noise sites. As monitoring interests expand to include low-energy urban sources only detectable close to the source, case studies are needed to demonstrate the challenges and benefits of urban infrasound monitoring. This case study highlights one approach to overcoming urban challenges and identifies a signal's source in a complex acoustic field. One 38 m and one 120 m aperture infrasound arrays were deployed on building rooftops north of downtown Dallas, Texas. Structural signals in the recorded data were identified, and the backazimuth to the source determined with frequency-wavenumber analysis. Fourteen days of data were analyzed to produce 314 coherent continuous-wave packets, with 246 of these detections associated with a narrow range of backazimuth directions. Analyzing the backazimuths from the two arrays identified the Mockingbird Bridge as the probable source which was the verified with seismic measurement on the structure. Techniques described here overcame the constraints imposed by urban environments and provide a basis to monitor infrastructure and its conditions at local distances (0-100 km).

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5.

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate 'occluded' state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

Reconstructing the transport cycle in the sugar porter superfamily using coevolution-powered machine learning.

Sugar porters (SPs) represent the largest group of secondary-active transporters. Some members, such as the glucose transporters (GLUTs), are well known for their role in maintaining blood glucose homeostasis in mammals, with their expression upregulated in many types of cancers. Because only a few sugar porter structures have been determined, mechanistic models have been constructed by piecing together structural states of distantly related proteins. Current GLUT transport models are predominantly descriptive and oversimplified. Here, we have combined coevolution analysis and comparative modeling, to predict structures of the entire sugar porter superfamily in each state of the transport cycle. We have analyzed the state-specific contacts inferred from coevolving residue pairs and shown how this information can be used to rapidly generate free-energy landscapes consistent with experimental estimates, as illustrated here for the mammalian fructose transporter GLUT5. By comparing many different sugar porter models and scrutinizing their sequence, we have been able to define the molecular determinants of the transport cycle, which are conserved throughout the sugar porter superfamily. We have also been able to highlight differences leading to the emergence of proton-coupling, validating, and extending the previously proposed latch mechanism. Our computational approach is transferable to any transporter, and to other protein families in general.

Establishing mammalian GLUT kinetics and lipid composition influences in a reconstituted-liposome system.

Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.

Structure of a mitochondrial ATP synthase with bound native cardiolipin.

The mitochondrial ATP synthase fuels eukaryotic cells with chemical energy. Here we report the cryo-EM structure of a divergent ATP synthase dimer from mitochondria of Euglena gracilis, a member of the phylum Euglenozoa that also includes human parasites. It features 29 different subunits, 8 of which are newly identified. The membrane region was determined to 2.8 Å resolution, enabling the identification of 37 associated lipids, including 25 cardiolipins, which provides insight into protein-lipid interactions and their functional roles. The rotor-stator interface comprises four membrane-embedded horizontal helices, including a distinct subunit a. The dimer interface is formed entirely by phylum-specific components, and a peripherally associated subcomplex contributes to the membrane curvature. The central and peripheral stalks directly interact with each other. Last, the ATPase inhibitory factor 1 (IF1) binds in a mode that is different from human, but conserved in Trypanosomatids.