Structure of the Myelin Sheath Proteolipid Plasmolipin (PLLP) in a Ganglioside-Containing Lipid Raft.

BACKGROUND: Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as neurological disorders, type 2 diabetes, and cancer metastasis. To better understand how PLLP interacts with raft components (gangliosides and cholesterol), we undertook a global study combining in silico simulations and physicochemical measurements of molecular interactions in various PLLP-ganglioside systems. METHODS: In silico studies consisted of molecular dynamics simulations in reconstructed membrane environments. PLLP-ganglioside interaction measurements were performed by microtensiometry at the water-air interface on ganglioside monolayers. RESULTS: We have elucidated the mode of interaction of PLLP with ganglioside GM1 and characterized this interaction at the molecular level. We showed that GM1 induces the structuring of the extracellular loops of PLLP and that this interaction propagates a conformational signal through the plasma membrane, involving a cholesterol molecule located between transmembrane domains. This conformational wave is finally transmitted to the intracellular domain of the protein, consistent with the role of PLLP in signal transduction. CONCLUSIONS: This study is a typical example of the epigenetic dimension of protein structure, a concept developed by our team to describe the chaperone effect of gangliosides on disordered protein motifs which associate with lipid rafts. From a physiological point of view, these data shed light on the role of gangliosides in myelin formation. From a pathological point of view, this study will help to design innovative therapeutic strategies focused on ganglioside-PLLP interactions in various PLLP-associated diseases.

Electrostatic Surface Potential as a Key Parameter in Virus Transmission and Evolution: How to Manage Future Virus Pandemics in the Post-COVID-19 Era.

Virus-cell interactions involve fundamental parameters that need to be considered in strategies implemented to control viral outbreaks. Among these, the surface electrostatic potential can give valuable information to deal with new epidemics. In this article, we describe the role of this key parameter in the hemagglutination of red blood cells and in the co-evolution of synaptic receptors and neurotransmitters. We then establish the functional link between lipid rafts and the electrostatic potential of viruses, with special emphasis on gangliosides, which are sialic-acid-containing, electronegatively charged plasma membrane components. We describe the common features of ganglioside binding domains, which include a wide variety of structures with little sequence homology but that possess key amino acids controlling ganglioside recognition. We analyze the role of the electrostatic potential in the transmission and intra-individual evolution of HIV-1 infections, including gatekeeper and co-receptor switch mechanisms. We show how to organize the epidemic surveillance of influenza viruses by focusing on mutations affecting the hemagglutinin surface potential. We demonstrate that the electrostatic surface potential, by modulating spike-ganglioside interactions, controls the hemagglutination properties of coronaviruses (SARS-CoV-1, MERS-CoV, and SARS-CoV-2) as well as the structural dynamics of SARS-CoV-2 evolution. We relate the broad-spectrum antiviral activity of repositioned molecules to their ability to disrupt virus-raft interactions, challenging the old concept that an antibiotic or anti-parasitic cannot also be an antiviral. We propose a new concept based on the analysis of the electrostatic surface potential to develop, in real time, therapeutic and vaccine strategies adapted to each new viral epidemic.

Structural basis of botulinum neurotoxin serotype A1 binding to human SV2A or SV2C receptors.

Botulinum neurotoxin A1 (BoNT/A1) is the most potent natural poison in human. BoNT/A1 recognize the luminal domain of SV2A (LD-SV2A) and its glycosylation at position N573 (N573g) or the luminal domain of SV2C (LD-SV2C) and its glycosylation at position N559 (N559g) to bind neural membrane. Our computational data suggest that the N-glycan at position 480 (N480g) in the luminal domain of SV2C (LD-SV2C) indirectly enhanced the contacts of the neurotoxin surface with the second N-glycan at position 559 (N559g) by acting as a shield to prevent N559g to interact with residues of LD-SV2C. The absence of an N-glycan homologous to N480g in LD-SV2A leads to a decrease of the binding of N573g to the surface of BoNT/A1. Concerning the intermolecular interactions between BoNT/A and the protein part of LD-SV2A or LD-SV2C, we showed that the high affinity of the neurotoxin for binding LD-SV2C are mediated by a better compaction of its F557-F562 part provided by a π-π network mediated by residues F547, F552, F557 and F562 coupled with the presence of two aromatic residues at position 563 and 564 that optimize the binding of BoNT/A1 via cation-pi and CH-pi interaction. Finally, in addition to the well-known ganglioside binding site which accommodates a ganglioside on the surface of BoNT/A1, we identified a structure we coined the ganglioside binding loop defined by the sequence 1253-HQFNNIAK-1260 that is conserved across all subtypes of BoNT/A and is predicted to has a high affinity to interact with gangliosides. These data solved the puzzle generated by mutational studies that could be only partially understood with crystallographic data that lack both a biologically relevant membrane environment and a full glycosylation of SV2.

AmyP53 Prevents the Formation of Neurotoxic ß-Amyloid Oligomers through an Unprecedent Mechanism of Interaction with Gangliosides: Insights for Alzheimer's Disease Therapy.

A broad range of data identify Ca2+-permeable amyloid pores as the most neurotoxic species of Alzheimer's ß-amyloid peptide (Aß1-42). Following the failures of clinical trials targeting amyloid plaques by immunotherapy, a consensus is gradually emerging to change the paradigm, the strategy, and the target to cure Alzheimer's disease. In this context, the therapeutic peptide AmyP53 was designed to prevent amyloid pore formation driven by lipid raft microdomains of the plasma membrane. Here, we show that AmyP53 outcompetes Aß1-42 binding to lipid rafts through a unique mode of interaction with gangliosides. Using a combination of cellular, physicochemical, and in silico approaches, we unraveled the mechanism of action of AmyP53 at the atomic, molecular, and cellular levels. Molecular dynamics simulations (MDS) indicated that AmyP53 rapidly adapts its conformation to gangliosides for an optimal interaction at the periphery of a lipid raft, where amyloid pore formation occurs. Hence, we define it as an adaptive peptide. Our results describe for the first time the kinetics of AmyP53 interaction with lipid raft gangliosides at the atomic level. Physicochemical studies and in silico simulations indicated that Aß1-42 cannot interact with lipid rafts in presence of AmyP53. These data demonstrated that AmyP53 prevents amyloid pore formation and cellular Ca2+ entry by competitive inhibition of Aß1-42 binding to lipid raft gangliosides. The molecular details of AmyP53 action revealed an unprecedent mechanism of interaction with lipid rafts, offering innovative therapeutic opportunities for lipid raft and ganglioside-associated diseases, including Alzheimer's, Parkinson's, and related proteinopathies.

Structural Basis of Botulinum Toxin Type F Binding to Glycosylated Human SV2A: In Silico Studies at the Periphery of a Lipid Raft.

Botulinum neurotoxins are the deadliest microbial neurotoxins in humans, with a lethal dose of 1 ng/kg. Incidentally, these neurotoxins are also widely used for medical and cosmetic purposes. However, little is known about the molecular mechanisms that control binding of botulinum neurotoxin type F1 (BoNT/F1) to its membrane receptor, glycosylated human synaptic vesicle glycoprotein A (hSV2Ag). To elucidate these mechanisms, we performed a molecular dynamics simulation (MDS) study of initial binding kinetics of BoNT/F1 to SV2A. Since this toxin also interacts with gangliosides, the simulations were performed at the periphery of a lipid raft in the presence of both SV2A and gangliosides. Our study suggested that interaction of BoNT/F1 with SV2A is exclusively mediated by N-glycan moiety of SV2A, which interacts with aromatic residues Y898, Y910, F946, Y1059 and H1273 of this toxin. Thus, in contrast with botulinum neurotoxin A1 (BoNT/A1), BoNT/F1 does not interact with protein content of SV2A. We attributed this incapability to a barrage effect exerted by neurotoxin residues Y1132, Q1133 and K1134, which prevent formation of long-lasting intermolecular hydrogen bonds. We also provided structural elements that suggest that BoNT/F1 uses the strategy of BoNT/A1 combined with the strategy of botulinum neurotoxin type E to bind N-glycan of its glycoprotein receptor. Overall, our study opened a gate for design of a universal inhibitor aimed at disrupting N-glycan-toxin interactions and for bioengineering of a BoNT/F1 protein that may be able to bind protein content of synaptic vesicle glycoprotein for therapeutic purposes.

The Epigenetic Dimension of Protein Structure Is an Intrinsic Weakness of the AlphaFold Program.

One of the most important lessons we have learned from sequencing the human genome is that not all proteins have a 3D structure. In fact, a large part of the human proteome is made up of intrinsically disordered proteins (IDPs) which can adopt multiple structures, and therefore, multiple functions, depending on the ligands with which they interact. Under these conditions, one can wonder about the value of algorithms developed for predicting the structure of proteins, in particular AlphaFold, an AI which claims to have solved the problem of protein structure. In a recent study, we highlighted a particular weakness of AlphaFold for membrane proteins. Based on this observation, we have proposed a paradigm, referred to as "Epigenetic Dimension of Protein Structure" (EDPS), which takes into account all environmental parameters that control the structure of a protein beyond the amino acid sequence (hence "epigenetic"). In this new study, we compare the reliability of the AlphaFold and Robetta algorithms' predictions for a new set of membrane proteins involved in human pathologies. We found that Robetta was generally more accurate than AlphaFold for ascribing a membrane-compatible topology. Raft lipids (e.g., gangliosides), which control the structural dynamics of membrane protein structure through chaperone effects, were identified as major actors of the EDPS paradigm. We conclude that the epigenetic dimension of a protein structure is an intrinsic weakness of AI-based protein structure prediction, especially AlphaFold, which warrants further development.

Molecular landscape of BoNT/B bound to a membrane-inserted synaptotagmin/ganglioside complex.

Botulinum neurotoxin serotype B (BoNT/B) uses two separate protein and polysialoglycolipid-binding pockets to interact with synaptotagmin 1/2 and gangliosides. However, an integrated model of BoNT/B bound to its neuronal receptors in a native membrane topology is still lacking. Using a panel of in silico and experimental approaches, we present here a new model for BoNT/B binding to neuronal membranes, in which the toxin binds to a preassembled synaptotagmin-ganglioside GT1b complex and a free ganglioside allowing a lipid-binding loop of BoNT/B to interact with the glycone part of the synaptotagmin-associated GT1b. Furthermore, our data provide molecular support for the decrease in BoNT/B sensitivity in Felidae that harbor the natural variant synaptotagmin2-N59Q. These results reveal multiple interactions of BoNT/B with gangliosides and support a novel paradigm in which a toxin recognizes a protein/ganglioside complex.

Structural Dynamics of the SARS-CoV-2 Spike Protein: A 2-Year Retrospective Analysis of SARS-CoV-2 Variants (from Alpha to Omicron) Reveals an Early Divergence between Conserved and Variable Epitopes.

We analyzed the epitope evolution of the spike protein in 1,860,489 SARS-CoV-2 genomes. The structural dynamics of these epitopes was determined by molecular modeling approaches. The D614G mutation, selected in the first months of the pandemic, is still present in currently circulating SARS-CoV-2 strains. This mutation facilitates the conformational change leading to the demasking of the ACE2 binding domain. D614G also abrogated the binding of facilitating antibodies to a linear epitope common to SARS-CoV-1 and SARS-CoV-2. The main neutralizing epitope of the N-terminal domain (NTD) of the spike protein showed extensive structural variability in SARS-CoV-2 variants, especially Delta and Omicron. This epitope is located on the flat surface of the NTD, a large electropositive area which binds to electronegatively charged lipid rafts of host cells. A facilitating epitope located on the lower part of the NTD appeared to be highly conserved among most SARS-CoV-2 variants, which may represent a risk of antibody-dependent enhancement (ADE). Overall, this retrospective analysis revealed an early divergence between conserved (facilitating) and variable (neutralizing) epitopes of the spike protein. These data aid in the designing of new antiviral strategies that could help to control COVID-19 infection by mimicking neutralizing antibodies or by blocking facilitating antibodies.

The epigenetic dimension of protein structure.

Accurate prediction of protein structure is one of the most challenging goals of biology. The most recent achievement is AlphaFold, a machine learning method that has claimed to have solved the structure of almost all human proteins. This technological breakthrough has been compared to the sequencing of the human genome. However, this triumphal statement should be treated with caution, as we identified serious flaws in some AlphaFold models. Disordered regions are often represented by large loops that clash with the overall protein geometry, leading to unrealistic structures, especially for membrane proteins. In fact, AlphaFold comes up against the notion that protein folding is not solely determined by genomic information. We suggest that all parameters controlling the structure of a protein without being strictly encoded in its amino acid sequence should be coined "epigenetic dimension of protein structure." Such parameters include for instance protein solvation by membrane lipids, or the structuration of disordered proteins upon ligand binding, but exclude sequence-encoded sites of post-translational modifications such as glycosylation. In our view, this paradigm is necessary to reconcile two opposite properties of living systems: beyond rigorous biological coding, evolution has given way to a certain level of uncertainty and anarchy.

Ganglioside binding domains in proteins: Physiological and pathological mechanisms.

Gangliosides are anionic lipids that form condensed membrane clusters (lipid rafts) and exert major regulatory functions on a wide range of proteins. In this review, we propose a new view of the structural features of gangliosides with special emphasis on emerging properties associated with protein binding modes. We analyze the different possibilities of molecular associations of gangliosides in lipid rafts and the role of cholesterol in this organization. We are particularly interested in amide groups of N-acetylated sugars which make it possible to neutralize the negative charge of the carboxylate group of sialic acids. We refer to this effect as "NH trick" and we demonstrate that it is operative in GM1, GD1a, GD1b and GT1b gangliosides. The NH trick is key to understand the different topologies adopted by gangliosides (chalice-like at the edge of lipid rafts, condensed clusters in central areas) and their impact on protein binding. We define three major types of ganglioside-binding domains (GBDs): α-helical, loop shaped, and large flat surface. We describe the mode of interaction of each GBD with typical reference proteins: synaptotagmin, 5HT1A receptor, cholera and botulinum toxins, HIV-1 surface envelope glycoprotein gp120, SARS-CoV-2 spike protein, cellular prion protein, Alzheimer's ß-amyloid peptide and Parkinson's disease associated α-synuclein. We discuss the common mechanisms and peculiarities of protein binding to gangliosides in the light of physiological and pathological conditions. We anticipate that innovative ganglioside-based therapies will soon show an exponential growth for the treatment of cancer, microbial infections, and neurodegenerative diseases.

Endocannabinoids Tune Intrinsic Excitability in O-LM Interneurons by Direct Modulation of Postsynaptic Kv7 Channels.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons, where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. Here, we show that Kv7 channel activity is upregulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons from rats of both sex, thus resulting in a synergistic long-term depression of intrinsic excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for induction. However, although LTD is dependent on cannabinoid type 1 receptors, LTD-IE is not. Molecular modeling shows a strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.SIGNIFICANCE STATEMENT In principal cells, Kv7 channels are essentially located at the axon initial segment. In contrast, in O-LM interneurons, Kv7 channels are highly expressed in the dendrites, suggesting a singular role of these channels in O-LM cell function. Here, we show that LTD of excitatory inputs in O-LM interneurons is associated with an upregulation of Kv7 channels, thus resulting in a synergistic LTD of LTD-IE. Both forms of plasticity are mediated by the biosynthesis of eCBs. Stimulation of CB1 receptors induces LTD, whereas the direct interaction of eCBs with Kv7 channels induces LTD-IE. Our results thus provide a previously unexpected involvement of eCBs in long-lasting plasticity of intrinsic excitability in GABAergic interneurons.

Structural dynamics of SARS-CoV-2 variants: A health monitoring strategy for anticipating Covid-19 outbreaks.

OBJECTIVES: the Covid-19 pandemic has been marked by sudden outbreaks of SARS-CoV-2 variants harboring mutations in both the N-terminal (NTD) and receptor binding (RBD) domains of the spike protein. The goal of this study was to predict the transmissibility of SARS-CoV-2 variants from genomic sequence data. METHODS: we used a target-based molecular modeling strategy combined with surface potential analysis of the NTD and RBD. RESULTS: we observed that both domains act synergistically to ensure optimal virus adhesion, which explains why most variants exhibit concomitant mutations in the RBD and in the NTD. Some mutation patterns affect the affinity of the spike protein for ACE-2. However, other patterns increase the electropositive surface of the spike, with determinant effects on the kinetics of virus adhesion to lipid raft gangliosides. Based on this new view of the structural dynamics of SARS-CoV-2 variants, we defined an index of transmissibility (T-index) calculated from kinetic and affinity parameters of coronavirus binding to host cells. The T-index is characteristic of each variant and predictive of its dissemination in animal and human populations. CONCLUSIONS: the T-index can be used as a health monitoring strategy to anticipate future Covid-19 outbreaks due to the emergence of variants of concern.