The 3D structure and mechanism of action are unknown for the integral plasma membrane transport protein solute carrier 4A10, which has been characterized functionally as an electroneutral Na+:HCO3- cotransporter. We used structure prediction and molecular dynamics simulations to study the binding of the transported ions to the solute carrier 4A10 protein and suggest a model of sequential binding of Na+ followed by HCO3- to the ion binding domain. The binding of HCO3- to the protein appears to depend absolutely on Na+ binding. Conversely, the binding of HCO3- stabilizes the interaction between Na+ and its binding site. Measurements of intracellular pH and Na+ concentration revealed the dependence of Na+ on HCO3- transport. The study lays the necessary foundation for targeted experimental analysis of ion translocation and for the development of selective transport inhibitors of solute carrier 4A10 and probably other proteins of the protein family of HCO3- transporters.

Hydrophobic interfaces are ubiquitous, and interactions of proteins with such surfaces remain an area of significant interest. α-Synuclein (α-Syn), a protein abundant in cerebrospinal fluid, is implicated in nearly 50 neurological disorders. The misfolding of α-Syn into amyloid aggregates is a critical step in the progression of several neurodegenerative diseases. Hydrophobic interfaces have been shown to catalyze this process. To better understand the mechanism of aggregation and ultimately aid in the development of therapeutic strategies, it is essential to probe the interfacial structures of α-Syn that may drive amyloid formation. Here, we present a detailed investigation of the interfacial structure of α-Syn on a polystyrene (PS) film, a widely used material in consumer products and laboratory settings. Elucidating the structural motifs of α-Syn at the PS interface provides insights into how plastic contaminants may induce conformational changes in the protein. Combining experimental vibrational sum frequency generation spectroscopy with theoretical spectral calculations, we identify the interfacial structure of α-Syn at the PS film and compare it with previously reported α-Syn conformation on air-water interfaces. The entire NAC region and majority of the residues in the N terminal are in direct contact with the PS surface, while the C terminal residues protrude away from the interface, staying in the solution. Our studies highlight the critical role of polymeric surfaces in facilitating α-Syn misfolding.

The impact of micro- and nanoplastics (MNPs) on human health is a growing field of research. Reports that MNPs can breach the blood-brain barrier and accumulate inside the brain have raised concerns over their possible involvement in the development of neurogenerative diseases. The aggregation of the abundant neuronal protein α-synuclein (α-syn) is pertinent to almost 50 neurological diseases including Parkinson's disease (PD). The role of nanoplastics in the formation of toxic aggregates is unclear and has been shown to depend strongly on the type of plastics. Here we report the molecular structure and orientation of human α-syn adsorbed on polystyrene NPs using interface-specific sum frequency scattering (SFS) and structure-sensitive two-dimensional infrared (2D IR) spectroscopy. The SFS experimental data were compared with the calculated spectra of several thousands of α-syn conformations generated from molecular dynamics simulations. The SFS results reveal that α-syn folds on polystyrene nanoplastics, adopting a partly helical structure with the N-terminus and nonamyloid component regions directly bound on the polystyrene nanosurface, while the C terminus protrudes away from the polystyrene interface. 2D IR results suggest that the entire α-syn corona comprises of partly aggregated α-syn structures, built of an ordered core enclosed with flexible dynamic regions. The data shed light on the mechanism by which α-syn folds and forms aggregates at the plastic particle surfaces, a link that has been missing in understanding the role of nanoplastic in the pathogenesis of PD and related neurodegenerative diseases.

Auxins are plant hormones that direct the growth and development of organisms on the basis of environmental cues. Indole-3-acetic acid (IAA) is the most abundant auxin in most plants. A variety of membrane transport proteins work together to distribute auxins. These include the AUX/LAX protein family that mediate auxin import from the apoplast to the cytosol. Here we use structural and biophysical approaches combined with molecular dynamics to study transport by Arabidopsis thaliana LAX3, which is essential for plant root formation. Transport assays document high-affinity transport of IAA, as well as competitive behaviour of the synthetic phenoxyacetic acid auxin herbicide 2,4-dichlorophenoxyacetic acid and the auxin transport inhibitors 1-naphthoxyacetic acid and 2-naphthoxyacetic acid. Four cryo-EM structures were solved with resolutions of 2.9–3.4 Å: an inward open apo structure, two inward semi-occluded structures in complex with IAA and 2,4-dichlorophenoxyacetic acid, and a fully occluded structure in complex with 2-naphthoxyacetic acid. Structurally, LAX3 consists of a bundle and a scaffold domain. The ligand-binding site is sandwiched between these domains with two histidines occupying positions analogous to the sodium-binding sites in distantly related sodium:neurotransmitter transporters. This architecture suggests that these histidines couple transport to the proton motive force. Molecular dynamics simulations are used to explore substrate binding and release, including their dependence on specific protonation states. This study advances our understanding of auxin recognition and transport by AUX/LAX, providing insights into a fundamental aspect of plant physiology and development. This study reveals the structural basis of auxin import by the AUX/LAX family. LAX3 binds auxin as well as herbicides via a proton-coupled mechanism, which offers insights into hormone recognition that is essential for lateral root growth.

Insulin binding to the insulin receptor (IR) induces large conformational changes leading to receptor activation. Although there exists a considerable number of IR structures in different conformational and insulin-saturation states, they cannot provide dynamic information or the resolved order of events leading to receptor activation. In this study, we employed molecular dynamics (MD) simulations to the experimentally solved structures of IR-insulin complexes occurring under physiological concentration conditions. We observed that insulin bound to the hybrid sites induced opening of site 1, and that site 1-bound insulin contributed to the extension of the α helix in the C terminus of the α chain (αCT) and increased inter-domain stabilization. Many models have previously been proposed for the activation of IR. Based on our observations, we propose a novel "ladder-climbing" mechanism of insulin-induced IR activation, where insulin gradually migrates from site 2 to site 1 while inducing a controlled conformational change in IR.

Current trends in molecular modeling are geared toward increasingly realistic representations of the biological environments reflected in larger, more complex systems. The complexity of the system-building procedure is ideally handled by software that converts user-provided descriptors into system coordinates. This, however, is not a trivial task, as building algorithms use simplifications that result in inaccuracies in the system properties. We created COBY, a coarse-grained system builder that can create a large variety of systems in a single command call with an improved accuracy of the complex membrane and solvent building procedures. COBY also contains features for building diverse systems in a single step, and has functionalities aiding force field development. COBY is an open-source software written in Python 3, and the code, documentation, and tutorials are hosted at https://github.com/MikkelDA/COBY.

Lipid membranes are central to cellular life. Complementing experiments, computational modeling has been essential in unraveling complex lipid-biomolecule interactions, crucial in both academia and industry. The Martini model, a coarse-grained force field for efficient molecular dynamics simulations, is widely used to study membrane phenomena but has faced limitations, particularly in capturing realistic lipid phase behavior. Here, we present refined Martini 3 lipid models with a mapping scheme that distinguishes lipid tails that differ by just two carbon atoms, enhancing the structural resolution and thermodynamic accuracy of model membrane systems including ternary mixtures. The expanded Martini lipid library includes thousands of models, enabling simulations of complex and biologically relevant systems. These advancements establish Martini as a robust platform for lipid-based simulations across diverse fields.

Permeability is a measure of the degree to which cells can transport molecules across biological barriers. Units of permeability are distance per unit time (typically cm/s), where accurate measurements are needed to define drug delivery in homeostasis and to model dysfunction occurring during disease. This perspective offers a set of community-led guidelines to benchmark permeability data across multidisciplinary approaches and different biological contexts. First, we lay out the analytical framework for three methodologies to calculate permeability: in silico assays using either transition-based counting or the inhomogeneous-solubility diffusion approaches, in vitro permeability assays using cells cultured in 2D or 3D geometries, and in vivo assays utilizing in situ brain perfusion or multiple time-point regression analysis. Then, we demonstrate a systematic benchmarking of in silico to both in vitro and in vivo, depicting the ways in which each benchmarking is sensitive to the choices of assay design. Finally, we outline seven recommendations for best practices in permeability benchmarking and underscore the significance of tailored permeability assays in driving advancements in drug delivery research and development. Our exploration encompasses a discussion of "generic" and tissue-specific biological barriers, including the blood-brain barrier (BBB), which is a major hurdle for the delivery of therapeutic agents into the brain. By addressing challenges in reconciling simulated data with experimental assays, we aim to provide insights essential for optimizing accuracy and reliability in permeability modeling.

Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.

Bufadienolides exert broad-spectrum pharmacological activities relevant to cardiology and novel cancer treatments. Their efficacy, toxicity, and pharmacokinetic profiles are significantly affected by modifications at carbon-3 (C3) of the steroid core. We have applied molecular dynamics simulations to characterize the consequences of (i) variations in size of the substituent at C3, (ii) the type of linker at C3 (ether vs. N-methoxy), and (iii) stereochemistry (C3β vs. C3α) for derivatives' interactions with Na+,K+-ATPase. The model compounds included bufalin, bufalin-N-glucose, bufalin-O-glucose as well as digoxigenin, digoxigenin monodigitoxoside and digoxin. It was shown that the optimal size of the substituent is a trade-off between the ability to form stabilizing interactions and steric and entropic interferences. The former is strongly affected by the nature of the linker due to its impact on the spatial position of the ligand: N-methoxy linker imposes rotational restrictions and places the core into a less favorable position compared to an ether bond. Similarly, the change from β- to α-anomer delocalizes the substituent precluding contacts with amino acid residues of the binding site. The presented mechanistic model of bufadienolide interactions with Na+,K+-ATPase helps to anticipate the consequences of modifications while designing derivatives with high anticancer activity but reduced cardiotoxicity.

The translation elongation factors eEF1A1 and eEF1A2 share 97% sequence similarity and perform similar roles in translation but exhibit mutually exclusive expression patterns in human tissues. Despite their high homology, they are linked to different diseases, likely due to paralog-specific interactions with distinct protein partners. The underlying reasons for these differences remain unclear. Here, using a combination of HDX-MS, MD, and SAXS approaches, we demonstrate that eEF1A1 and eEF1A2 exhibit distinct structural dynamics, leading to different structural organizations. eEF1A2 is a compact, stably folded protein, whereas eEF1A1 adopts multiple conformational states, including the opening and closing of the conformational space between domains D1 and D3, as well as significant internal and external dynamics of domain D2. These dynamics facilitate protein dimerization in eEF1A1, contrasting with eEF1A2, which apparently remains monomeric in solution, challenging previous X-ray crystallography findings. These data provide molecular insight into the functional differences between the highly homologous translation factors eEF1A1 and eEF1A2, potentially explaining their paralog-specific nontranslational roles and distinct contributions to human diseases.

Sortilin is a single-pass transmembrane receptor involved in intracellular trafficking, neurotrophic signaling, and protein clearance pathways relevant to neurodegenerative disease. We recently identified the neuron-specific protein NSG1 as a selective modulator of sortilin function, promoting its ectodomain shedding via ADAM10. However, the molecular basis of this interaction remains unresolved. Here, we present a structural framework for NSG1-mediated regulation of sortilin shedding. Using mutagenesis, biochemical assays, and structural modeling, we mapped the interaction interface of NSG1 to the helical transmembrane domain (TMD) of sortilin. We show that NSG1 binds a specific interface within the sortilin TMD, modulating its susceptibility to ectodomain shedding. Mutational analysis revealed that substitutions in the central region of the sortilin TMD, particularly T770W and A773W, significantly reduce NSG1-dependent shedding without disrupting complex formation. Coarse-grained molecular dynamics simulations identified two potential binding interfaces on the sortilin TMD and demonstrated that the T770W mutation shifts the preferred interface, thereby diminishing the ability of NSG1 to promote proteolytic processing. Notably, the closely related protein NSG2 has a different preferred binding mode on the sortilin TMD and does not induce shedding, highlighting the functional specificity of NSG1. Our findings establish the TMD-TMD interaction as an important basis for NSG1-mediated regulation of sortilin shedding. This study advances our understanding of how transmembrane interactions govern substrate-specific a disintegrin and metalloproteinase proteolysis and provides new insight into the molecular control of sortilin function. Given the emerging role of sortilin in Alzheimer's disease, these insights may help clarify how its processing is regulated in the diseased brain.

Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug's molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.

Current trends in molecular modelling are geared towards increasingly realistic representations of the modelled systems. This is reflected in larger, more complex systems, which are difficult to build and would ideally rely on a software that converts userprovided descriptors into system coordinates. This is not a trivial task, as the building algorithms use simplifications that can introduce inaccuracies in the system properties that do not correspond to the requested values. We created COBY, a coarse-grained system builder that can create a large variety of systems in a single command call using Martini molecule models. We improved the accuracy of the complex membrane and solvent building procedures, introduced a variety of arguments that can be used to build diverse systems, and implemented features intended for force field development. COBY can be used to build flat membranes of any degree of complexity, handle protein and solvent insertion, solute flooding, stacked membranes, membrane patches and pores, and includes advanced functionalities such as molecule import, lipid building from fragments, handling of multiple parameter libraries, and several choices of algorithms for interpreting user-provided system descriptors. COBY is an open-source software written in Python 3, and the code, documentation, and tutorials are hosted at github.com/MikkelDA/COBY. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

Sortilin is a cell surface receptor abundantly expressed in the central nervous system, and its primary function involves the trafficking and sorting of proteins. Sortilin is a transmembrane (TM) protein consisting of a large ectodomain, the Vps10p domain, an α-helix, anchoring the protein to the membrane, and an unstructured C-terminus. In a process called ectodomain shedding, sheddases can cleave the Vps10p domain off, causing secretases to cleave the TM region, which generates aggregates of C-terminal fragments.