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Because of high mutation rates, viruses constantly adapt to new environments. When propagated in cell lines, certain viruses acquire positively charged amino acids on their surface proteins, enabling them to utilize negatively charged heparan sulfate (HS) as an attachment receptor. In this study, we used enterovirus A71 (EV-A71) as the model and demonstrated that, unlike the parental MP4 variant, the cell-adapted strong HS-binder MP4-97R/167 G does not require acidification for uncoating and releases its genome in the neutral or weakly acidic environment of early endosomes. We experimentally confirmed that this pH-independent entry is not associated with the use of HS as an attachment receptor but rather with compromised capsid stability. We then extended these findings to another HS-dependent strain. In summary, our data indicate that the acquisition of capsid mutations conferring affinity for HS comes together with decreased capsid stability and allows EV-A71 to enter the cell via a pH-independent pathway. This pH-independent entry mechanism boosts viral replication in cell lines but may prove deleterious in vivo , especially for enteric viruses crossing the acidic gastric environment before reaching their primary replication site, the intestine. Our study thus provides new insight into the mechanisms underlying the in vivo attenuation of HS-binding EV-A71 strains. Not only are these viruses hindered in tissues rich in HS due to viral trapping, as generally accepted, but our research reveals that their diminished capsid stability further contributes to attenuation in vivo . This underscores the complex relationship between HS-binding, capsid stability, and viral fitness, where increased replication in cell lines coincides with attenuation in harsh in vivo environments like the gastrointestinal tract., Competing Interests: HT, SC, AM, GM, VC, MD, AA, SC, CT No competing interests declared, (© 2024, Tee et al.)

β-lactamases from Gram-negative bacteria are generally regarded as soluble, periplasmic enzymes. NDMs have been exceptionally characterized as lipoproteins anchored to the outer membrane. A bioinformatics study on all sequenced β-lactamases was performed that revealed a predominance of putative lipidated enzymes in the Class D OXAs. Namely, 60% of the OXA Class D enzymes contain a lipobox sequence in their signal peptide, that is expected to trigger lipidation and membrane anchoring. This contrasts with β-lactamases from other classes, which are predicted to be mostly soluble proteins. Almost all (>99%) putative lipidated OXAs are present in Acinetobacter spp. Importantly, we further demonstrate that OXA-23 and OXA-24/40 are lipidated, membrane-bound proteins in Acinetobacter baumannii . In contrast, OXA-48 (commonly produced by Enterobacterales) lacks a lipobox and is a soluble protein. Outer membrane vesicles (OMVs) from A. baumannii cells expressing OXA-23 and OXA-24/40 contain these enzymes in their active form. Moreover, OXA-loaded OMVs were able to protect A. baumannii , Escherichia coli , and Pseudomonas aeruginosa cells susceptible to piperacillin and imipenem. These results permit us to conclude that membrane binding is a bacterial host-specific phenomenon in OXA enzymes. These findings reveal that membrane-bound β-lactamases are more common than expected and support the hypothesis that OMVs loaded with lipidated β-lactamases are vehicles for antimicrobial resistance and its dissemination. This advantage could be crucial in polymicrobial infections, in which Acinetobacter spp. are usually involved, and underscore the relevance of identifying the cellular localization of lactamases to better understand their physiology and target them.IMPORTANCEβ-lactamases represent the main mechanism of antimicrobial resistance in Gram-negative pathogens. Their catalytic function (cleaving β-lactam antibiotics) occurs in the bacterial periplasm, where they are commonly reported as soluble proteins. A bioinformatic analysis reveals a significant number of putative lipidated β-lactamases, expected to be attached to the outer bacterial membrane. Notably, 60% of Class D OXA β-lactamases (all from Acinetobacter spp.) are predicted as membrane-anchored proteins. We demonstrate that two clinically relevant carbapenemases, OXA-23 and OXA-24/40, are membrane-bound proteins in A. baumannii . This cellular localization favors the secretion of these enzymes into outer membrane vesicles that transport them outside the boundaries of the cell. β-lactamase-loaded vesicles can protect populations of antibiotic-susceptible bacteria, enabling them to thrive in the presence of β-lactam antibiotics. The ubiquity of this phenomenon suggests that it may have influenced the dissemination of resistance mediated by Acinetobacter spp., particularly in polymicrobial infections, being a potent evolutionary advantage.

MolecularWebXR is a new web-based platform for education, science communication and scientific peer discussion in chemistry and biology, based on modern web-based Virtual Reality (VR) and Augmented Reality (AR). With no installs as it is all web-served, MolecularWebXR enables multiple users to simultaneously explore, communicate and discuss concepts about chemistry and biology in immersive 3D environments, by manipulating and passing around objects with their bare hands and pointing at different elements with natural hand gestures. Users may either be present in the same physical space or distributed around the world, in the latter case talking naturally with each other thanks to built-in audio. While MolecularWebXR offers the most immersive experience on high-end AR/VR headsets, its WebXR core also supports participation on consumer devices such as smartphones (with optional cardboard goggles for enhanced immersion), computers, and tablets. MolecularWebXR includes preset VR rooms covering topics in general, inorganic, and organic chemistry, as well as biophysics, structural biology, and general biology. Users can also add new content via the PDB2AR tool. We demonstrate MolecularWebXR's versatility and ease of use across a wide age range (12-80) in fully virtual and mixed real-virtual sessions at science outreach events, undergraduate and graduate courses, scientific collaborations, and conference presentations. MolecularWebXR is available for free use without registration at https://molecularwebxr.org. A blog post version of this preprint with embedded videos is available at https://go.epfl.ch/molecularwebxr-blog-post., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Aerolysin is a bacterial toxin that forms transmembrane pores at the host plasma membrane and has a narrow internal diameter and great stability. These assets make it a highly promising nanopore for detecting biopolymers such as nucleic acids and peptides. Although much is known about aerolysin from a microbiological and structural perspective, its membrane association and pore-formation mechanism are not yet fully understood. Here, we used angle-resolved second harmonic scattering (AR-SHS) and single-channel current measurements to investigate how wild-type (wt) aerolysin and its mutants interact with liposomes in aqueous solutions at femtomolar concentrations. Our AR-SHS experiments were sensitive enough to detect changes in the electrostatic properties of membrane-bound aerolysin, which were induced by variations in pH levels. We reported for the first time the membrane binding affinity of aerolysin at different stages of the pore formation mechanism: while wt aerolysin has a binding affinity as high as 20 fM, the quasi-pore and the prepore states show gradually decreasing membrane affinities, incomplete insertion, and a pore opening signature. Moreover, we quantitatively characterized the membrane affinity of mutants relevant for applications to nanopore sensing. Our study provides a label-free method for efficiently screening biological pores suitable for conducting molecular sensing and sequencing measurements as well as for probing pore-forming processes.

Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have described patients with deletions and nonsense variants in PREPL, but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.

Protein design and engineering are evolving at an unprecedented pace leveraging the advances in deep learning. Current models nonetheless cannot natively consider non-protein entities within the design process. Here, we introduce a deep learning approach based solely on a geometric transformer of atomic coordinates and element names that predicts protein sequences from backbone scaffolds aware of the restraints imposed by diverse molecular environments. To validate the method, we show that it can produce highly thermostable, catalytically active enzymes with high success rates. This concept is anticipated to improve the versatility of protein design pipelines for crafting desired functions., (© 2024. The Author(s).)

Protein-ligand interactions (PLIs) determine the efficacy and safety profiles of small molecule drugs. Existing methods rely on either structural information or resource-intensive computations to predict PLI, casting doubt on whether it is possible to perform structure-free PLI predictions at low computational cost. Here we show that a light-weight graph neural network (GNN), trained with quantitative PLIs of a small number of proteins and ligands, is able to predict the strength of unseen PLIs. The model has no direct access to structural information about the protein-ligand complexes. Instead, the predictive power is provided by encoding the entire chemical and proteomic space in a single heterogeneous graph, encapsulating primary protein sequence, gene expression, the protein-protein interaction network, and structural similarities between ligands. This novel approach performs competitively with, or better than, structure-aware models. Our results suggest that existing PLI prediction methods may be improved by incorporating representation learning techniques that embed biological and chemical knowledge., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jitao David Zhang, Simon J. Crouzet, Anja Maria Lieberherr, Kenneth Atz, Tobias Nilsson, Lisa Sach- Peltason and Alex T. Müller report financial support, administrative support and article publishing charges were provided by 10.13039/100004337F. Hoffmann-La Roche Ltd. Jitao David Zhang, Kenneth Atz, Tobias Nilsson, Lisa Sach-Peltason, Alex T. Müller report being currently employed by F Hoffmann-La Roche Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Huntington's disease is a neurodegenerative disorder caused by an expanded polyglutamine stretch near the N-terminus of the huntingtin (HTT) protein, rendering the protein more prone to aggregate. The first 17 residues in HTT (Nt17) interact with lipid membranes and harbor multiple post-translational modifications (PTMs) that can modulate HTT conformation and aggregation. In this study, we used a combination of biophysical studies and molecular simulations to investigate the effect of PTMs on the helicity of Nt17 in the presence of various lipid membranes. We demonstrate that anionic lipids such as PI4P, PI(4,5)P2, and GM1 significantly enhance the helical structure of unmodified Nt17. This effect is attenuated by single acetylation events at K6, K9, or K15, whereas tri-acetylation at these sites abolishes Nt17-membrane interaction. Similarly, single phosphorylation at S13 and S16 decreased but did not abolish the POPG and PIP2-induced helicity, while dual phosphorylation at these sites markedly diminished Nt17 helicity, regardless of lipid composition. The helicity of Nt17 with phosphorylation at T3 is insensitive to the membrane environment. Oxidation at M8 variably affects membrane-induced helicity, highlighting a lipid-dependent modulation of the Nt17 structure. Altogether, our findings reveal differential effects of PTMs and crosstalks between PTMs on membrane interaction and conformation of HTT. Intriguingly, the effects of phosphorylation at T3 or single acetylation at K6, K9, and K15 on Nt17 conformation in the presence of certain membranes do not mirror that observed in the absence of membranes. Our studies provide novel insights into the complex relationship between Nt17 structure, PTMs, and membrane binding.

Cardiolipin (CL) is a mitochondria-specific phospholipid that forms heterotypic interactions with membrane-shaping proteins and regulates the dynamic remodeling and function of mitochondria. However, the precise mechanisms through which CL influences mitochondrial morphology are not well understood. In this study, employing molecular dynamics (MD) simulations, we observed CL localize near the membrane-binding sites of the mitochondrial fusion protein Optic Atrophy 1 (OPA1). To validate these findings experimentally, we developed a bromine-labeled CL probe to enhance cryoEM contrast and characterize the structure of OPA1 assemblies bound to the CL-brominated lipid bilayers. Our images provide direct evidence of interactions between CL and two conserved motifs within the paddle domain (PD) of OPA1, which control membrane-shaping mechanisms. We further observed a decrease in membrane remodeling activity for OPA1 in lipid compositions with increasing concentrations of monolyso-cardiolipin (MLCL). Suggesting that the partial replacement of CL by MLCL accumulation, as observed in Barth syndrome-associated mutations of the tafazzin phospholipid transacylase, compromises the stability of protein-membrane interactions. Our analyses provide insights into how biological membranes regulate the mechanisms governing mitochondrial homeostasis., Competing Interests: Competing Interest Statement A.F. and F.R.M. are shareholders and employees of Altos Labs.

7An efficient immune system must provide protection against a broad range of pathogens without causing excessive collateral tissue damage. While immune effectors have been well characterized, we know less about the resilience mechanisms protecting the host from its own immune response. Antimicrobial peptides (AMPs) are small, cationic peptides that contribute to innate defenses by targeting negatively charged membranes of microbes. While protective against pathogens, AMPs can be cytotoxic to host cells. Here, we reveal that a family of stress-induced proteins, the Turandots, protect the Drosophila respiratory system from AMPs, increasing resilience to stress. Flies lacking Turandot genes are susceptible to environmental stresses due to AMP-induced tracheal apoptosis. Turandot proteins bind to host cell membranes and mask negatively charged phospholipids, protecting them from cationic pore-forming AMPs. Collectively, these data demonstrate that Turandot stress proteins mitigate AMP cytotoxicity to host tissues and therefore improve their efficacy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

Organ laterality of vertebrates is specified by accelerated asymmetric decay of Dand5 mRNA mediated by Bicaudal-C1 (Bicc1) on the left side, but whether binding of this or any other mRNA to Bicc1 can be regulated is unknown. Here, we found that a CRISPR-engineered truncation in ankyrin and sterile alpha motif (SAM)-containing 3 (ANKS3) leads to symmetric mRNA decay mediated by the Bicc1-interacting Dand5 3' UTR. AlphaFold structure predictions of protein complexes and their biochemical validation by in vitro reconstitution reveal a novel interaction of the C-terminal coiled coil domain of ANKS3 with Bicc1 that inhibits binding of target mRNAs, depending on the conformation of ANKS3 and its regulation by ANKS6. The dual regulation of RNA binding by mutually opposing structured protein domains in this multivalent protein network emerges as a novel mechanism linking associated laterality defects and possibly other ciliopathies to perturbed dynamics in Bicc1 ribonucleoparticle (RNP) formation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Rothé et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Distinct morphologies of the mitochondrial network support divergent metabolic and regulatory processes that determine cell function and fate 1-3 . The mechanochemical GTPase optic atrophy 1 (OPA1) influences the architecture of cristae and catalyses the fusion of the mitochondrial inner membrane 4,5 . Despite its fundamental importance, the molecular mechanisms by which OPA1 modulates mitochondrial morphology are unclear. Here, using a combination of cellular and structural analyses, we illuminate the molecular mechanisms that are key to OPA1-dependent membrane remodelling and fusion. Human OPA1 embeds itself into cardiolipin-containing membranes through a lipid-binding paddle domain. A conserved loop within the paddle domain inserts deeply into the bilayer, further stabilizing the interactions with cardiolipin-enriched membranes. OPA1 dimerization through the paddle domain promotes the helical assembly of a flexible OPA1 lattice on the membrane, which drives mitochondrial fusion in cells. Moreover, the membrane-bending OPA1 oligomer undergoes conformational changes that pull the membrane-inserting loop out of the outer leaflet and contribute to the mechanics of membrane remodelling. Our findings provide a structural framework for understanding how human OPA1 shapes mitochondrial morphology and show us how human disease mutations compromise OPA1 functions., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Proteins are essential molecular building blocks of life, responsible for most biological functions as a result of their specific molecular interactions. However, predicting their  binding  interfaces remains a challenge. In this study, we present a geometric transformer that acts directly on atomic coordinates labeled only with element names. The resulting model-the Protein Structure Transformer, PeSTo-surpasses the current state of the art in predicting protein-protein interfaces and can also predict and differentiate between interfaces involving nucleic acids, lipids, ions, and small molecules with high confidence. Its low computational cost enables processing high volumes of structural data, such as molecular dynamics ensembles allowing for the discovery of interfaces that remain otherwise inconspicuous in static experimentally solved structures. Moreover, the growing foldome provided by de novo structural predictions can be easily analyzed, providing new opportunities to uncover unexplored biology., (© 2023. The Author(s).)

Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ., Competing Interests: Declaration of interests J.J.C. is a consultant for Thermo Scientific. A.F. is a shareholder and employee of Altos Labs and a shareholder and consultant for Relay Therapeutics, LLC., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Several groups developed in the last years augmented and virtual reality (AR/VR) software to visualize 3D molecules, most rather static, limited in content, and requiring software installs, some even requiring expensive hardware. We launched in 2020 moleculARweb (https://molecularweb.epfl.ch), a website that offers interactive content for chemistry and structural biology education through commodity web-based AR that works on consumer devices like smartphones, tablets and laptops. Among thousands of users, teachers increasingly request more biological macromolecules to be available, a demand that we cannot address individually. Therefore, to allow users to build their own material, we built a web interface where they can create online AR experiences in few steps starting from Protein Data Bank, AlphaFold or custom uploaded structures, or from virtual objects/scenes exported from the Visual Molecular Dynamics program, without any programming knowledge. The web tool also returns WebXR sessions for viewing and manipulating the models in WebXR-compatible devices including smartphones, tablets, and also immersive VR headsets with WebXR-capable browsers, where models can be manipulated even with bare hands when supported by the device. The tool is accessible for free at https://molecularweb.epfl.ch/pages/pdb2ar.html., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Evolution has found countless ways to transport material across cells and cellular compartments separated by membranes. Protein assemblies are the cornerstone for the formation of channels and pores that enable this regulated passage of molecules in and out of cells, contributing to maintaining most of the fundamental processes that sustain living organisms. As in several other occasions, we have borrowed from the natural properties of these biological systems to push technology forward and have been able to hijack these nano-scale proteinaceous pores to learn about the physical and chemical features of molecules passing through them. Today, a large repertoire of biological pores is exploited as molecular sensors for characterizing biomolecules that are relevant for the advancement of life sciences and application to medicine. Although the technology has quickly matured to enable nucleic acid sensing with transformative implications for genomics, biological pores stand as some of the most promising candidates to drive the next developments in single-molecule proteomics., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

moleculARweb (https://molecularweb.epfl.ch) began as a website for education and outreach in chemistry and structural biology through augmented reality (AR) content that runs in the web browsers of regular devices like smartphones, tablets, and computers. Here we present two evolutions of moleculARweb's Virtual Modeling Kits (VMK), tools where users can build and view molecules, and explore their mechanics, in 3D AR by handling the molecules in full 3D with custom-printed cube markers (VMK 2.0) or by moving around a simulated scene with mouse or touch gestures (VMK 3.0). Upon simulation the molecules experience visually realistic torsions, clashes, and hydrogen-bonding interactions that the user can manually switch on and off to explore their effects. Moreover, by manually tuning a fictitious temperature the users can accelerate conformational transitions or 'freeze' specific conformations for careful inspection in 3D. Even some phase transitions and separations can be simulated. We here showcase these and other features of the new VMKs connecting them to possible specific applications to teaching and self-learning of concepts from general, organic, biological and physical chemistry; and in assisting with small tasks in molecular modelling for research. Last, in a short discussion section we overview what future developments are needed for the 'dream tool' for the future of chemistry education and work., (Copyright 2022 Fabio Cortes Rodriguez, Lucien Krapp, Matteo Dal Peraro, Luciano Abriata. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin-5 for fusion of endoplasmic reticulum-derived vesicles with the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER-to-Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild-type, among them ERGIC-53. The BET1/ERGIC-53 interaction was validated by endogenous co-immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC-53 was observed in P1 and P2's derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC-53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)