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To develop more sensitive fluorescent protein sensors for nicotine, we combined computational protein design, site-saturated, site-directed, and combinatorial mutagenesis with fluorescence assays, molecular dynamics simulations, and absorbance measurements. The data showed that the resulting molecules, iNicSnFR11 and iNicSnFR12, have higher sensitivity to nicotine than previously reported constructs. In the linear portion of the dose-response relation at sub-μM [nicotine] for iNicSnFR12, ∆F/F0increased with a proportionality constant (S-slope) of 2.6 μM-1, representing a 6.5-fold higher sensitivity than iNicSnFR3a. Molecular dynamics calculations enabled identification of a binding pose for nicotine previously indeterminate from experimental data. Further comparative simulations based on this model revealed a tilt in helix 4 in the optimized sensor, likely altering allosteric networks involving the ligand binding site. The absorbance data showed that the fluorescence activation results from increased absorption rather than increased quantum yield for fluorescence. iNicSnFR12 resolved nicotine in diluted mouse and human serum at the peak concentration (100-200 nM) that occurs during smoking or vaping, but also at the decaying concentrations (< 100 nM) during the intervals between smoking or vaping sessions. NicSnFR12 was roughly as sensitive to varenicline or acetylcholine as to nicotine; the sensitivity to choline was at least one order of magnitude less. None of these drugs would markedly distort measurements in human biofluids such as sweat and interstitial fluid. Therefore, iNicSnFR12 is a promising candidate as the molecular sensor that could underlie a continuous nicotine monitor for human biofluids.

Molecular dynamics simulations of the alpha-7 nicotinic receptor, both atomistic (ion permeation free energy calculation) and coarse-grained (lipid and drug binding)., Two amino residues were incorrectly assigned in the original PDB file. N35 should be S35; P111 should be S111. However, given they are neither inside the TMD region---affecting the lipid binding---nor facing toward to pore---affecting the ion permeation, the results in this manuscript remain intact. Control experiments have been performed.

Ligand-gated ion channels transduce electrochemical signals in neurons and other excitable cells. Aside from canonical ligands, phospholipids are thought to bind specifically to the transmembrane domain of several ion channels. However, structural details of such lipid contacts remain elusive, partly due to limited resolution of these regions in experimental structures. Here, we discovered multiple lipid interactions in the channel GLIC by integrating cryo-electron microscopy and large-scale molecular simulations. We identified 25 bound lipids in the GLIC closed state, a conformation where none, to our knowledge, were previously known. Three lipids were associated with each subunit in the inner leaflet, including a buried interaction disrupted in mutant simulations. In the outer leaflet, two intrasubunit sites were evident in both closed and open states, while a putative intersubunit site was preferred in open-state simulations. This work offers molecular details of GLIC-lipid contacts particularly in the ill-characterized closed state, testable hypotheses for state-dependent binding, and a multidisciplinary strategy for modeling protein-lipid interactions.

Voltage gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive, and that a conformational change involving a transition to aπhelix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel fromAliarcobacter butzleri(NavAb), with characteristic pore hydration, ion permeation and drug binding properties. Furthermore, we show that aπ-helix transition can lead to pore opening, and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for aπ-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important model sodium channel.

Pentameric ligand-gated ion channels (pLGICs) perform electrochemical signal transduction in organisms ranging from bacteria to humans. Among the prokaryotic pLGICs, there is architectural diversity involving N-terminal domains (NTDs) not found in eukaryotic relatives, exemplified by the calcium-sensitive channel (DeCLIC) from a Desulfofustis deltaproteobacterium, which has an NTD in addition to the canonical pLGIC structure. Here, we have characterized the structure and dynamics of DeCLIC through cryoelectron microscopy (cryo-EM), small-angle neutron scattering (SANS), and molecular dynamics (MD) simulations. In the presence and absence of calcium, cryo-EM yielded structures with alternative conformations of the calcium-binding site. SANS profiles further revealed conformational diversity at room temperature beyond that observed in static structures, shown through MD to be largely attributable to rigid-body motions of the NTD relative to the protein core, with expanded and asymmetric conformations improving the fit of the SANS data. This work reveals the range of motion available to the DeCLIC NTD and calcium-binding site, expanding the conformational landscape of the pLGIC family. Further, these findings demonstrate the power of combining low-resolution scattering, high-resolution structural, and MD simulation data to elucidate interfacial interactions that are highly conserved in the pLGIC family.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Mangrove forest encroachment into coastal marsh habitats has been described in subtropical regions worldwide in recent decades. To better understand how soil processes may influence vegetation change, we studied soil surface elevation change, accretion rates, and soil subsurface change across a coastal salinity gradient in Florida, USA, an area with documented mangrove encroachment into saline marshes. Our aim was to identify if variations in the soil variables studied exist and to document any associated vegetation shifts. We used surface elevation tables and marker horizons to document the soil variables over 5 years in a mangrove-to-marsh transition zone or ecotone. Study sites were located in three marsh types (brackish, salt, and transition) and in riverine mangrove forests. Mangrove forest sites had significantly higher accretion rates than marsh sites and were the only locations where elevation gain occurred. Significant loss in surface elevation occurred at transition and salt marsh sites. Transition marshes, which had a significantly higher rate of shallow subsidence compared to other wetland types, appear to be most vulnerable to submergence or to a shift to mangrove forest. Submergence can result in herbaceous vegetation mortality and conversion to open water, with severe implications to the quantity and quality of wetland services provided.

Worldwide, human activities have modified hydrology and nutrient loading regimes in coastal wetlands. Understanding the interplay between these drivers and subsequent response of wetland plant communities is essential to informing wetland management and restoration efforts. Recent restoration strategies in Louisiana proposes to use sediment diversions from the Mississippi River to build land in adjacent wetlands and reduce the rate of land to open water conversion. In conjunction with sediment delivery, diversions can increase nutrient loads and water levels in the receiving basins. We conducted a greenhouse mesocosm experiment in which we exposed three common tidal freshwater and brackish marsh plants (Panicum hemitomon, Sagittaria lancifolia, and Spartina patens) to two nitrate loading rates [high (35 g N m2 year−1) and low (0.25 g N m2 year−1)], and two flooding treatments (with and without diversion pulsing). Experimental units were set at two different elevations within the treatment tanks to simulate both a healthy and degraded marsh. Plant growth metrics and soil physicochemical properties were measured monthly. Final total biomass was determined at the study’s conclusion. Growth responses differed between species but were not significantly influenced by the treatments. Soil redox potential decreased significantly following the increase in flooding associated with the diversion pulse, but recovered to pre-diversion levels after a 3-month recovery period. Our study suggests short flooding pulses with a recovery period may be key for maintaining healthy marshes, however there remains a need for longer-term empirical studies to understand marsh response to pressures associated with river sediment diversions over time.

A goal of wetland restoration is the establishment of resilient plant communities that persist under a variety of environmental conditions. We investigated the role of intraspecific and interspecific variation on plant community establishment in a brackish marsh that had been restored by sediment addition. Plant growth, sediment accretion, and surface elevation change in planted, not-planted, and nearby reference sites (treatments) were compared. Four perennial macrophytes were planted: Bolboschoenus robustus, Distichlis spicata, Phragmites australis, and Schoenoplectus californicus. There was 100% survival of the planted species, and all exhibited rapid vegetative spread. Intraspecific variation in stem height and cover was identified, and interspecific comparisons also indicated differences in species cover. Treatment comparisons revealed that final total cover at not-planted sites was equivalent to that at reference sites, and was highest at planted sites where P. australis became dominant. Species richness was initially highest at the reference sites, but final richness was equivalent among treatments. Soil surface elevation was greater at planted compared to not-planted and reference sites. Because of the rapid cover and increased surface elevation generated by planted species, the resiliency of restored coastal marshes may be enhanced by plantings in areas where natural colonization is slow and subsidence is high.

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.

Cryo-EM structures and molecular simulations of a pentameric ligand-gated ion channel, GLIC, in resting and activating conditions implicate protonation sites and altered dynamics in sequential gating., Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1–β2 and M2–M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction in various organisms from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural bases for specific pLGIC-lipid interactions remain poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and binding and modulation by lipids, offering a simplified model system for structure-function relationship studies. In this study, functional effects of noncanonical amino acid substitution of a potential lipid-interacting residue (W206) at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid-binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206, consistent with cation-π binding. Polar contacts from other regions of the protein, particularly M3 residue Q264, further support lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor ε subunit, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ε-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, suggesting a potential role of this site in channel gating. In conclusion, this work details type-specific lipid interactions, which adds to our growing understanding of how lipids modulate pLGICs. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:297 issue:2 ispartof: location:United States status: published

SARS-CoV-2 contains four structural proteins in its genome. These proteins aid in the assembly and budding of new virions at the ER-Golgi intermediate compartment (ERGIC). Current fundamental research efforts largely focus on one of these proteins – the spike (S) protein. Since successful antiviral therapies are likely to target multiple viral components, there is considerable interest in understanding the biophysical role of its other structural proteins, in particular structural membrane proteins. Here, we have focused our efforts on the characterization of the full-length envelope (E) protein from SARS-CoV-2, combining experimental and computational approaches. Recombinant expression of the full-length E protein from SARS-CoV-2 reveals that this membrane protein is capable of independent multimerization, possibly as a tetrameric or smaller species. Fluorescence microscopy shows that the protein localizes intracellularly, and coarse-grained MD simulations indicate it causes bending of the surrounding lipid bilayer, corroborating a potential role for the E protein in viral budding. Although we did not find robust electrophysiological evidence of ion-channel activity, cells transfected with the E protein exhibited reduced intracellular Ca2+, which may further promote viral replication. However, our atomistic MD simulations revealed that previous NMR structures are relatively unstable, and result in models incapable of ion conduction. Our study highlights the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insights, and eventually permitting virtual drug screening.

Pentameric ligand-gated ion channels (pLGICs) are crucial mediators of electrochemical signal transduction from bacteria to humans. Lipids play an important role in regulating pLGIC function, yet the structural basis for specific pLGIC-lipid interactions remains poorly understood. The bacterial channel ELIC recapitulates several properties of eukaryotic pLGICs, including activation by the neurotransmitter GABA and sensitivity to lipids, offering a simplified model system for structure-function studies. In this study, functional effects of non-canonical amino acid substitution of W206 at the top of the M1-helix, combined with detergent interactions observed in recent X-ray structures, are consistent with this region being the location of a lipid binding site on the outward face of the ELIC transmembrane domain. Coarse-grained and atomistic molecular dynamics simulations revealed preferential binding of lipids containing a positive charge, particularly involving interactions with residue W206 consistent with cation-π binding. Polar contacts from the principal subunit, particularly M3 residue Q264, further supported lipid binding via headgroup ester linkages. Aromatic residues were identified at analogous sites in a handful of eukaryotic family members, including the human GABAA receptor subunit ɛ, suggesting conservation of relevant interactions in other evolutionary branches. Further mutagenesis experiments indicated that mutations at this site in ɛ-containing GABAA receptors can change the apparent affinity of the agonist response to GABA, consistent with a potential role of this site in channel gating. In conclusion, this work is a detailed case study in type-specific lipid interactions at an evolutionarily distinctive pLGIC site, with implications for lipid modulation and lipophilic drug design.

AbstractRT-LAMP detection of SARS-CoV-2 has been shown as a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validated the optimized RT-LAMP assay for the detection of SARS-CoV-2 in raw nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings with limited economic resources.

The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation remain unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components.

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological development in this family relies on high-quality structural data in multiple, subtly distinct functional states. However, structural data remain limited, particularly for the unliganded or resting state. Here we report cryo-electron microscopy structures of the Gloeobacter violaceus ligand-gated ion channel (GLIC) under resting and activating conditions (neutral and low pH). Parallel models were built either manually or using recently developed density-guided molecular simulations. The moderate resolution of resting-state reconstructions, particularly in the extracellular domain, was improved under activating conditions, enabling the visualization of residues at key subunit interfaces including loops B, C, F, and M2–M3. Combined with molecular dynamics simulations, the cryo-electron microscopy structures at different pH describe a heterogeneous population of closed channels, with activating conditions condensing the closed-channel energy landscape on a pathway towards gating.

Pentameric ligand-gated ion channels (pLGICs) are allosteric receptors that mediate rapid electrochemical signal transduction in the animal nervous system through the opening of an ion pore upon binding of neurotransmitters. Orthologs have been found and characterized in prokaryotes and they display highly similar structure–function relationships to eukaryotic pLGICs; however, they often encode greater architectural diversity involving additional amino-terminal domains (NTDs). Here we report structural, functional, and normal-mode analysis of two conformational states of a multidomain pLGIC, called DeCLIC, from a Desulfofustis deltaproteobacterium, including a periplasmic NTD fused to the conventional ligand-binding domain (LBD). X-ray structure determination revealed an NTD consisting of two jelly-roll domains interacting across each subunit interface. Binding of Ca 2+ at the LBD subunit interface was associated with a closed transmembrane pore, with resolved monovalent cations intracellular to the hydrophobic gate. Accordingly, DeCLIC-injected oocytes conducted currents only upon depletion of extracellular Ca 2+ ; these were insensitive to quaternary ammonium block. Furthermore, DeCLIC crystallized in the absence of Ca 2+ with a wide-open pore and remodeled periplasmic domains, including increased contacts between the NTD and classic LBD agonist-binding sites. Functional, structural, and dynamical properties of DeCLIC paralleled those of sTeLIC, a pLGIC from another symbiotic prokaryote. Based on these DeCLIC structures, we would reclassify the previous structure of bacterial ELIC (the first high-resolution structure of a pLGIC) as a “locally closed” conformation. Taken together, structures of DeCLIC in multiple conformations illustrate dramatic conformational state transitions and diverse regulatory mechanisms available to ion channels in pLGICs, particularly involving Ca 2+ modulation and periplasmic NTDs.

Increasing atmospheric carbon dioxide (CO2) concentrations are likely to influence future distributions of plants and plant community structure in many regions of the world through effects on photosynthetic rates. In recent decades the encroachment of woody mangrove species into herbaceous marshes has been documented along the U.S. northern Gulf of Mexico coast. These species shifts have been attributed primarily to rising sea levels and warming winter temperatures, but the role of elevated CO2 and water availability may become more prominent drivers of species interactions under future climate conditions. Drought has been implicated as a major factor contributing to salt marsh vegetation dieback in this region. In this greenhouse study we examined the effects of CO2 concentration (∼380 ppm, ∼700 ppm) and water regime (drought, saturated, flooded) on early growth of Avicennia germinans, a C3 mangrove species, and Spartina alterniflora, a C4 grass. Plants were grown in monocultures and in a mixed-species assemblage. We found that neither species responded to elevated CO2 over the 10-month duration of the experiment, and there were few interactions between experimental factors. Two effects of water regime were documented: lower A. germinans pneumatophore biomass under drought conditions, and lower belowground biomass under flooded conditions regardless of planting assemblage. Evidence of interspecific interactions was noted. Competition for aboveground resources (e.g., light) was indicated by lower S. alterniflora stem biomass in mixed-species assemblage compared to biomass in S. alterniflora monocultures. Pneumatophore biomass of A. germinans was reduced when grown in monoculture compared to the mixed-species assemblage, indicating competition for belowground resources. These interactions provide insight into how these species may respond following major disturbance events that lead to vegetation dieback. Site variation in propagule availability and physico-chemical conditions will determine plant community composition and structure following such disturbances when these two species co-occur.

Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

The superfamily of pentameric ligand-gated ion channels (pLGICs) comprises key players in electrochemical signal transduction across evolution, including historic model systems for receptor allostery and targets for drug development. Accordingly, structural studies of these channels have steadily increased, and now approach 250 depositions in the protein data bank. This review contextualizes currently available structures in the pLGIC family, focusing on morphology, ligand binding, and gating in three model subfamilies: the prokaryotic channel GLIC, the cation-selective nicotinic acetylcholine receptor, and the anion-selective glycine receptor. Common themes include the challenging process of capturing and annotating channels in distinct functional states; partially conserved gating mechanisms, including remodeling at the extracellular/transmembrane-domain interface; and diversity beyond the protein level, arising from posttranslational modifications, ligands, lipids, and signaling partners. Interpreting pLGIC structures can be compared to describing an elephant in the dark, relying on touch alone to comprehend the many parts of a monumental beast: each structure represents a snapshot in time under specific experimental conditions, which must be integrated with further structure, function, and simulations data to build a comprehensive model, and understand how one channel may fundamentally differ from another.

Mangrove forests are highly productive tidal saline wetland ecosystems found along sheltered tropical and subtropical coasts. Ecologists have long assumed that climatic drivers (i.e., temperature and rainfall regimes) govern the global distribution, structure, and function of mangrove forests. However, data constraints have hindered the quantification of direct climate–mangrove linkages in many parts of the world. Recently, the quality and availability of global-scale climate and mangrove data have been improving. Here, we used these data to better understand the influence of air temperature and rainfall regimes upon the distribution, abundance, and species richness of mangrove forests. Although our analyses identify global-scale relationships and thresholds, we show that the influence of climatic drivers is best characterized via regional range-limit-specific analyses. We quantified climatic controls across targeted gradients in temperature and/or rainfall within 14 mangrove distributional range limits. Climatic thresholds for mangrove presence, abundance, and species richness differed among the 14 studied range limits. We identified minimum temperature-based thresholds for range limits in eastern North America, eastern Australia, New Zealand, eastern Asia, eastern South America, and southeast Africa. We identified rainfall-based thresholds for range limits in western North America, western Gulf of Mexico, western South America, western Australia, Middle East, northwest Africa, east central Africa, and west-central Africa. Our results show that in certain range limits (e.g., eastern North America, western Gulf of Mexico, eastern Asia), winter air temperature extremes play an especially important role. We conclude that rainfall and temperature regimes are both important in western North America, western Gulf of Mexico, and western Australia. With climate change, alterations in temperature and rainfall regimes will affect the global distribution, abundance, and diversity of mangrove forests. In general, warmer winter temperatures are expected to allow mangroves to expand poleward at the expense of salt marshes. However, dispersal and habitat availability constraints may hinder expansion near certain range limits. Along arid and semiarid coasts, decreases or increases in rainfall are expected to lead to mangrove contraction or expansion, respectively. Collectively, our analyses quantify climate–mangrove linkages and improve our understanding of the expected global- and regional-scale effects of climate change upon mangrove forests.

Cryo-EM Structure Determination and Model Fitting of the Proton-Gated Ligand-Gated Ion Channel glic at Multiple pH States