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B cell translocation gene 1 (BTG1) protein belongs to the BTG/transducer of ERBB2 (TOB) family of antiproliferative proteins whose members regulate various key cellular processes such as cell cycle progression, apoptosis, and differentiation. Somatic missense mutations in BTG1 are found in ∼70% of a particularly malignant and disseminated subtype of diffuse large B cell lymphoma (DLBCL). Antiproliferative activity of BTG1 has been linked to its ability to associate with transcriptional cofactors and various enzymes. However, molecular mechanisms underlying these functional interactions and how the disease-linked mutations in BTG1 affect these mechanisms are currently unknown. To start filling these knowledge gaps, here, using atomistic molecular dynamics (MD) simulations, we explored structural, dynamic, and kinetic characteristics of BTG1 protein, and studied how various DLBCL mutations affect these characteristics. We focused on the protein region formed by α2 and α4 helices, as this interface has been reported not only to serve as a binding hotspot for several cellular partners but also to harbor sites for the majority of known DLBCL mutations. Markov state modeling analysis of extensive MD simulations revealed that the α2-α4 interface in the wild-type (WT) BTG1 undergoes conformational transitions between closed and open metastable states. Importantly, we show that some of the mutations in this region that are observed in DLBCL, such as Q36H, F40C, Q45P, E50K (in α2), and A83T and A84E (in α4), either overstabilize one of these two metastable states or give rise to new conformations in which these helices are distorted (i.e., kinked or unfolded). Based on these results, we conclude that the rapid interconversion between the closed and open conformations of the α2-α4 interface is an essential component of the BTG1 functional dynamics that can prime the protein for functional associations with its binding partners. Disruption of the native dynamic equilibrium by DLBCL mutants leads to the ensemble of conformations in BTG1 that are unlikely structurally and/or kinetically to enable productive functional interactions with the binding proteins.

Molecular dynamics (MD) simulations have become increasingly impactful in membrane biophysics because they offer atomistic resolution into the atomistic fluctuations of lipid assemblies. Validation of the simulation trajectories with experimental data is crucial for interpretation and application of MD results. As an ideal benchmarking technique, NMR spectroscopy delivers order parameters of the carbon−deuterium bond fluctuations along the lipid chains. Additionally, NMR relaxation can access lipid dynamics providing yet another point for validation of simulation force fields. Here we performed short resampling simulations of membrane trajectories to investigate the lipid CH bond fluctuations on sub-40-ps timescales to explore the local fast dynamics. We recently established a robust framework for analysis of NMR relaxation rates from MD simulations, which improves upon current approaches and shows excellent agreement of experimental and theoretical results. The calculation of relaxation rates from simulations presents a universal challenge that we addressed by hypothesizing the existence of fast CH bond dynamics that evade the analysis of simulation data with temporal resolution of 40 ps (or lower). Indeed, our results support this hypothesis confirming the validity of our solution to the sampling problem. Furthermore, we show that the fast CH bond dynamics occur on timescales at which carbon−carbon bond conformations appear nearly stationary and unaffected by cholesterol. Lastly, we discuss the correspondence to the CH bond dynamics of liquid hydrocarbons and relate their existence to the apparent microviscosity of the bilayer hydrocarbon core.STATEMENT OF SIGNIFICANCENuclear magnetic resonance data have been historically used to validate membrane simulations through the average order parameters of the lipid chains. However, the bond dynamics that give rise to this equilibrium bilayer structure have rarely been compared between in vitro and in silico systems despite the availability of substantial experimental data. Here we investigate the logarithmic timescales sampled by the lipid chain motions and confirm a recently developed computational protocol that creates a dynamics-based bridge between simulations and NMR spectroscopy. Our results establish the foundations for validating a relatively unexplored dimension of bilayer behavior and thus have far-reaching applications in membrane biophysics.

Rapid flip-flop of phospholipids across the two leaflets of biological membranes is crucial for many aspects of cellular life. The transport proteins that facilitate this process are classified as pump-like flippases and floppases and channel-like scramblases. Unexpectedly, Class A G protein–coupled receptors (GPCRs), a large class of signaling proteins exemplified by the visual receptor rhodopsin and its apoprotein opsin, are constitutively active as scramblases in vitro. In liposomes, opsin scrambles lipids at a unitary rate of >100,000 per second. Atomistic molecular dynamics simulations of opsin in a lipid membrane reveal conformational transitions that expose a polar groove between transmembrane helices 6 and 7. This groove enables transbilayer lipid movement, conceptualized as the swiping of a credit card (lipid) through a card reader (GPCR). Conformational changes that facilitate scrambling are distinct from those associated with GPCR signaling. In this review, we discuss the physiological significance of GPCR scramblase activity and the modes of its regulation in cells.

Abnormal aggregation of the Amyloid β (Aβ) peptides into fibrils play a critical role in the development of Alzheimer’s disease. A two-stage “dock-lock” model have been proposed for the Aβ fibril elongation process. However, the mechanisms of the Aβ monomer-fibril binding process have not been elucidated with the necessary molecular-level precision, and so it remains unclear how the lock phase dynamics leads to the overall in-register binding of the Aβ monomer onto the fibril. To gain mechanistic insight into this critical step during the fibril elongation process we used molecular dynamics (MD) simulations with a physics-based coarse-grained UNited-RESidue (UNRES) force field we sampled extensively the dynamics of the lock phase process, in which a fibril-bound Aβ((9–40)) peptide rearranges to establish the native docking conformation. Analysis of the MD trajectories with Markov State Models was used to quantify the kinetics of the rearrangement process and the most probable pathways leading to the overall native docking conformation of the incoming peptide. These revealed a key intermediate state in which an intra-monomer hairpin is formed between the central core amyloidogenic patch (18)VFFA(21) and the C-terminal hydrophobic patch (34)LMVG(37). This hairpin structure is highly favored as a transition state during the lock phase of the fibril elongation. We propose a molecular mechanism for the facilitation of the Aβ fibril elongation by amyloidogenic hydrophobic patches.

Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Lipid bilayers form the main matrix of functional cell membranes, and their dynamics underlie a host of physical and biological processes. Here we show that elastic membrane properties and collective molecular dynamics are related by the mean-square amplitudes (order parameters) and relaxation rates (correlation times) of lipid acyl chain motions. We performed all-atom molecular dynamics simulations of liquid-crystalline bilayers to further interpret available NMR data. Our analysis entailed development of a theoretical framework that allows direct comparison of carbon-hydrogen (CH) bond relaxations as measured by simulations and NMR experiments. The new formalism enables validation of lipid force fields against NMR data by including a fixed bilayer normal (director axis) and restricted anisotropic motion of the CH bonds described by their segmental order parameters. The simulated spectral density of thermally excited CH bond fluctuations exhibited well-defined spin-lattice (Zeeman) relaxations analogous to those measured by solid-state NMR spectroscopy. Their frequency signature could be fit to a simple power-law function, indicative of collective dynamics of a nematic-like nature. The calculated spin-lattice relaxation rates scaled as the squared order parameters of the lipid acyl chains yielding an apparent bending modulus κC of the bilayer. Our results show a strong correlation with κC values obtained from solid-state NMR studies of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers with varying amounts of cholesterol as further validated by neutron spin-echo measurements of membrane elasticity. The simulations uncover a critical role of interleaflet coupling in membrane mechanics and thus provide insights into the molecular sites of emerging elastic properties within lipid bilayers.STATEMENT OF SIGNIFICANCEThe lipid make-up of a bilayer determines its measurable properties but how the motions of individual molecules combine to produce these properties remains unclear. By exploiting the synergy between NMR spectroscopy and molecular dynamics (MD) simulations, we show that the lipid dynamics in a bilayer are collective yet segmental in nature and contribute directly to bilayer elasticity. Comparison between MD simulations and NMR entails an improved theoretical framework that allows the two techniques to be directly related. This study thus provides novel insights into the inner workings of lipid membranes while delivering a new tool for validating computational approaches against experimental data.

Understanding structural dynamics of biomolecules at the single-molecule level is vital to advancing our knowledge of molecular mechanisms. Currently, there are few techniques that can capture dynamics at the sub-nanometre scale and in physiologically relevant conditions. Atomic force microscopy (AFM)1 has the advantage of analysing unlabelled single molecules in physiological buffer and at ambient temperature and pressure, but its resolution limits the assessment of conformational details of biomolecules2. Here we present localization AFM (LAFM), a technique developed to overcome current resolution limitations. By applying localization image reconstruction algorithms3 to peak positions in high-speed AFM and conventional AFM data, we increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions. LAFM enables the calculation of high-resolution maps from either images of many molecules or many images of a single molecule acquired over time, facilitating single-molecule structural analysis. LAFM is a post-acquisition image reconstruction method that can be applied to any biomolecular AFM dataset.

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Lipid nanodiscs are small synthetic lipid bilayer structures that are stabilized in solution by special circumscribing (or scaffolding) proteins or polymers. Because they create native-like environments for transmembrane proteins, lipid nanodiscs have become a powerful tool for structural determination of this class of systems when combined with cryo-electron microscopy or nuclear magnetic resonance. The elastic properties of lipid bilayers determine how the lipid environment responds to membrane protein perturbations, and how the lipid in turn modifies the conformational state of the embedded protein. However, despite the abundant use of nanodiscs in determining membrane protein structure, the elastic material properties of even pure lipid nanodiscs (i.e., without embedded proteins) have not yet been quantitatively investigated. A major hurdle is due to the inherently nonlocal treatment of the elastic properties of lipid systems implemented by most existing methods, both experimental and computational. In addition, these methods are best suited for very large “infinite” size lipidic assemblies, or ones that contain periodicity, in the case of simulations. We have previously described a computational analysis of molecular dynamics simulations designed to overcome these limitations, so it allows quantification of the bending rigidity (KC) and tilt modulus (κt) on a local scale even for finite, nonperiodic systems, such as lipid nanodiscs. Here we use this computational approach to extract values of KC and κt for a set of lipid nanodisc systems that vary in size and lipid composition. We find that the material properties of lipid nanodiscs are different from those of infinite bilayers of corresponding lipid composition, highlighting the effect of nanodisc confinement. Nanodiscs tend to show higher stiffness than their corresponding macroscopic bilayers, and moreover, their material properties vary spatially within them. For small-size MSP1 nanodiscs, the stiffness decreases radially, from a value that is larger in their center than the moduli of the corresponding bilayers by a factor of ∼2–3. The larger nanodiscs (MSP1E3D1 and MSP2N2) show milder spatial changes of moduli that are composition dependent and can be maximal in the center or at some distance from it. These trends in moduli correlate with spatially varying structural properties, including the area per lipid and the nanodisc thickness. Finally, as has previously been reported, nanodiscs tend to show deformations from perfectly flat circular geometries to varying degrees, depending on size and lipid composition. The modulations of lipid elastic properties that we find should be carefully considered when making structural and functional inferences concerning embedded proteins.

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain-containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P

In coronaviruses, the fusion peptide (FP) is situated within the membrane fusion domain of the spike protein, becoming exposed following proteolytic cleavages at the S1/S2 and S2’ sites. After receptor binding-induced conformational changes, the FP penetrates the host cell membrane and mediates membrane fusion. Previous work has revealed the importance of calcium for SARS-CoV-1 FP structural stability and host membrane insertion. In this follow-up study, we systematically introduced charge-neutralizing alanine mutations in the negatively charged amino acids within the SARS-CoV-1 fusion peptide (E801A, D802A, D812A, E821A, D825A, D830A) to identify residues that likely bind to calcium. We assayed fusion competency by performing a syncytia-formation assay in VeroE6 cells and infectivity using pseudoparticles. The loss of single negatively charged residues D812 or D830 greatly reduced syncytia formation and produced noninfectious pseudoparticles. Furthermore, we observed a calcium-dependent decrease in the infectivity of the D825A and D830A pseudoparticles, as well as fewer syncytia in the cells expressing E821A/D825A double mutant FP. To clarify which residue pairs in the FP are most likely to bind calcium and promote host membrane insertion, we carried out molecular dynamics (MD) simulations of the various FP constructs. From our modeling, residue E801 is predicted to pair with either D830 or D802 to coordinate one calcium; a second calcium ion likely pairs residue E821 with either D812 or D825. We propose a model of bimodal calcium binding in the FP1 and FP2 domains, which anchors the SARS-CoV-1 FP in the host cell membrane to promote membrane insertion and fusion.

Recent discoveries about functional mechanisms of proteins in the TMEM16 family of phospholipid scramblases have illuminated the dual role of the membrane as both the substrate and a mechanistically responsive environment in the wide range of physiological processes and genetic disorders in which they are implicated. This is highlighted in the review of recent findings from our collaborative investigations of molecular mechanisms of TMEM16 scramblases that emerged from iterative functional, structural, and computational experimentation. In the context of this review, we present new MD simulations and trajectory analyses motivated by the fact that new structural information about the TMEM16 scramblases is emerging from cryo-EM determinations in lipid nanodiscs. Because the functional environment of these proteins in in vivo and in in vitro is closer to flat membranes, we studied comparatively the responses of the membrane to the TMEM16 proteins in flat membranes and nanodiscs. We find that bilayer shapes in the nanodiscs are very different from those observed in the flat membrane systems, but the function-related slanting of the membrane observed at the nhTMEM16 boundary with the protein is similar in the nanodiscs and in the flat bilayers. This changes, however, in the bilayer composed of longer-tail lipids, which is thicker near the phospholipid translocation pathway, which may reflect an enhanced tendency of the long tails to penetrate the pathway and create, as shown previously, a nonconductive environment. These findings support the correspondence between the mechanistic involvement of the lipid environment in the flat membranes, and the nanodiscs. © 2019 Wiley Periodicals, Inc.

Both lipid and ion translocation by Ca2+-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein structures, but how these conformations form, and what functions they support, remains unknown. From analyses of atomistic molecular dynamics simulations of Ca2+-bound nhTMEM16 we find that the mechanism of a conformational transition of the groove from membrane-exposed to occluded from the membrane involves the repositioning of transmembrane helix 4 (TM4) following its disengagement from a TM3/TM4 interaction interface. Residue L302 is a key element in the hydrophobic TM3/TM4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryogenic electron microscopy (cryo-EM) reveals a partially closed groove that could translocate ions, but not lipids. This is corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A., A membrane-exposed groove in Ca2+-gated TMEM16 scramblases forms the translocation pathway for ions and lipids. Here authors combine molecular dynamics with cryo-EM and functional assays to uncover the conformational transitions of the groove leading to a non-selective ion channel pore.

Because lipid bilayers can bend and stretch in ways similar to thin elastic sheets, physical models of bilayer deformation have utilized mechanical constants such as the moduli for bending rigidity (κC) and area compressibility (KA). However, the use of these models to quantify the energetics of membrane deformation associated with protein-membrane interactions and the membrane response to stress is often hampered by the shortage of experimental data suitable for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations can provide alternative means to estimateKAvalues, current approaches suffer significant technical limitations. Here, we present a novel computational framework that allows for a direct estimation ofKAvalues for individual bilayer leaflets. The theory is based on the concept of elasticity and derivesKAfrom real-space analysis of local thickness fluctuations sampled in MD simulations. We explore and validate the model on a large set of single and multicomponent bilayers of different lipid composition and sizes, simulated at different temperatures. The calculated bilayer compressibility moduli agree with values estimated previously from experiments and those obtained from a standard computational method based on a series of constrained tension simulations. We further validate our framework in a comparison with an existing polymer brush model (PBM) and confirm the PBM’s predicted linear relationship with proportionality coefficient of 24 using elastic parameters calculated from the simulation trajectories. The robustness of the results that emerge from the new method allows us to revisit the origins of the bilayer mechanical (compressible) thickness and in particular, its dependence on acyl chain unsaturation and the presence of cholesterol.

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources1–6. This nutrient is transported across the blood–brain and blood–retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner7,8. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents. Cryo-electron microscopy and molecular dynamics simulations reveal how MFSD2A transports essential omega-3 fatty acids across the blood–brain and blood–retina barriers as lysolipids.

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.STATEMENT OF SIGNIFICANCESARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed “fusion peptide” (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

Neurotransmitter:sodium symporters (NSS) are conserved from bacteria to man and serve as targets for drugs, including antidepressants and psychostimulants. Here we report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation. To obtain this structure, we were guided by findings from single-molecule fluorescence spectroscopy and molecular dynamics simulations indicating that L-Phe binding and mutation of the conserved N-terminal Trp8 to Ala both promote an inward-facing state. Compared to the outward-facing occluded conformation, our structure reveals a major tilting of the cytoplasmic end of transmembrane segment (TM) 5, which, together with release of the N-terminus but without coupled movement of TM1, opens a wide cavity towards the second Na+ binding site. The structure of this key intermediate in the LeuT transport cycle, in the context of other NSS structures, leads to the proposal of an intracellular release mechanism of substrate and ions in NSS proteins., Neurotransmitter:sodium symporters (NSS) serve as targets for drugs including antidepressants and psychostimulants. Here authors report the X-ray structure of the prokaryotic NSS member, LeuT, in a Na+/substrate-bound, inward-facing occluded conformation which is a key intermediate in the LeuT transport cycle.

G protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new 3D molecular structures of GPCRs (3D-GPCRome) during the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. While experimentally-resolved structures undoubtedly provide valuable snapshots of specific GPCR conformational states, they give only limited information on their flexibility and dynamics associated with function. Molecular dynamics (MD) simulations have become a widely established technique to explore the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations requires efficient storage resources and specialized software, hence limiting the dissemination of these data to specialists in the field. Here we present the GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyse and share GPCR MD data. GPCRmd originates from a community-driven effort to create the first open, interactive, and standardized database of GPCR MD simulations. We demonstrate the power of this resource by performing comparative analyses of multiple GPCR simulations on two mechanisms critical to receptor function: internal water networks and sodium ion interaction.

G-protein-coupled receptors (GPCRs) are involved in numerous physiological processes and are the most frequent targets of approved drugs. The explosion in the number of new three-dimensional (3D) molecular structures of GPCRs (3D-GPCRome) over the last decade has greatly advanced the mechanistic understanding and drug design opportunities for this protein family. Molecular dynamics (MD) simulations have become a widely established technique for exploring the conformational landscape of proteins at an atomic level. However, the analysis and visualization of MD simulations require efficient storage resources and specialized software. Here we present GPCRmd (http://gpcrmd.org/), an online platform that incorporates web-based visualization capabilities as well as a comprehensive and user-friendly analysis toolbox that allows scientists from different disciplines to visualize, analyze and share GPCR MD data. GPCRmd originates from a community-driven effort to create an open, interactive and standardized database of GPCR MD simulations.

Members of the TMEM16/ANO family of membrane proteins are Ca2+-activated phospholipid scramblases and/or Cl− channels. A membrane-exposed hydrophilic groove in these proteins serves as a shared translocation pathway for ions and lipids. However, the mechanism by which lipids gain access to and permeate through the groove remains poorly understood. Here, we combine quantitative scrambling assays and molecular dynamic simulations to identify the key steps regulating lipid movement through the groove. Lipid scrambling is limited by two constrictions defined by evolutionarily conserved charged and polar residues, one extracellular and the other near the membrane mid-point. The region between these constrictions is inaccessible to lipids and water molecules, suggesting that the groove is in a non-conductive conformation. A sequence of lipid-triggered reorganizations of interactions between these residues and the permeating lipids propagates from the extracellular entryway to the central constriction, allowing the groove to open and coordinate the headgroups of transiting lipids., Some TMEM16 family members are Ca2+-dependent phospholipid scramblases, which also mediate non-selective ion transport; however, the mechanism how lipids permeate through the TMEM16 remains poorly understood. Here, the authors combine biochemical assays and simulations to identify the key steps regulating lipid movement through the membrane-exposed groove.

Neurotransmitter:sodium symporters (NSS), targets of antidepressants and psychostimulants, clear neurotransmitters from the synaptic cleft through sodium (Na+)-coupled transport. Substrate and Na+ are thought to be transported from the extracellular to intracellular space through an alternating access mechanism by coordinated conformational rearrangements in the symporter that alternately expose the binding sites to each side of the membrane. However, the mechanism by which the binding of ligands coordinates conformational changes occurring on opposite sides of the membrane is not well understood. Here, we report the use of single-molecule fluorescence resonance energy transfer (smFRET) techniques to image transitions between distinct conformational states on both the extracellular and intracellular sides of the prokaryotic NSS LeuT, including partially open intermediates associated with transport activity. The nature and functional context of these hitherto unidentified intermediate states shed new light on the allosteric mechanism that couples substrate and Na+ symport by the NSS family through conformational dynamics.

Allostery plays a crucial role in the mechanism of neurotransmitter-sodium symporters, such as the human dopamine transporter. To investigate the molecular mechanism that couples the transport-associated inward release of the Na+ ion from the Na2 site to intracellular gating, we applied a combination of the thermodynamic coupling function (TCF) formalism and Markov state model analysis to a 50-μs data set of molecular dynamics trajectories of the human dopamine transporter, in which multiple spontaneous Na+ release events were observed. Our TCF approach reveals a complex landscape of thermodynamic coupling between Na+ release and inward-opening, and identifies diverse, yet well-defined roles for different Na+-coordinating residues. In particular, we identify a prominent role in the allosteric coupling for the Na+-coordinating residue D421, where mutation has previously been associated with neurological disorders. Our results highlight the power of the TCF analysis to elucidate the molecular mechanism of complex allosteric processes in large biomolecular systems.

Somatic missense mutations of BTG1 are exclusive to germinal center (GC)-derived B cell lymphomas (~12% of DLBCLs) and are most prevalent in ABC-DLBCL (p=0.0184 vs GCB-DLBCL), particularly in the MCD/cluster 5 subtype, which features extranodal dissemination and unfavorable outcome. However, the relevance, mechanism of action and biological contribution of BTG1 mutations have not been studied. Using a rigorous genomic covariate analysis, we identified BTG1 mutations as a top genetic driver in DLBCL. Furthermore, molecular dynamics simulations indicated that BTG1 recurrent mutations, including the most frequent Q36H, disrupted the protein structure, with likely deleterious functional consequences. To investigate the effect of BTG1 mutation in GC B cells, we generated a conditional Btg1Q36H knock in mouse crossed to the B cell specific Cd19Cre line. Surprisingly, there was no apparent phenotype in GC B cells or other B cell populations. However, placing Btg1 Q36H and WT GC B cells in competition within the same mouse through adoptive transfer revealed a dramatic competitive advantage of Btg1 Q36H cells, virtually taking over the GC reaction. To gain further insight into this striking fitness advantage, we performed RNAseq in Btg1 Q36H GCs, which showed marked enrichment for genes induced in positively selected GC B cells, including MYC targets and biosynthetic pathways. The same genes were also enriched in BTG1 mutant DLBCL patients in 2 independent cohorts. Furthermore, Btg1 Q36H GC B cells displayed greater RNA content and cell size, reflecting increased fitness. Positive selection normally triggers a brief Myc pulse in GC B cells. We therefore crossed our Btg1Q36H mice to the MycGFPprotein fusion reporter and observed higher proportion of Myc GFP+ cells in Btg1 Q36H GCs. For mechanistic studies, we generated isogenic BTG1 Q36H or BTG1 WT human DLBCL cell lines. BTG1 Q36H cells exhibited enrichment for the same positively selected GC B and MYC target genes, as well as greater RNA content and cell size. BTG1 family members were suggested to interact with RNA. Performing RNA-immunoprecipitation, we discovered that ~800 transcripts associated with BTG1 WT, but not BTG1 Q36H. Notably, these corresponded to the same positively selected GC B and MYC target genes, including MYC itself. BTG1 was shown to regulate mRNA stability in other cell types. However, BTG1 Q36H did not alter MYC mRNA stability and instead facilitated MYC protein synthesis, thus disrupting a novel GC context-specific checkpoint mechanism, whereby BTG1 normally attenuates spurious MYC translation to tightly restrict fitness potential. In GC B cells, Myc induction coincides with S phase entry, but G2/M progression requires re-entry into the proliferative dark zone. To characterize GC dynamics in vivo, we performed targeted single cell RNAseq in competing Btg1 Q36H and WT GC B cells and noted earlier and higher proportion of positively selected Btg1 Q36H GC B cells having committed to G2/M and the proliferative program. We confirmed faster S phase completion in competing Btg1 Q36H GC B cells by in vivo EdU/BrdU labelling and greater re-entry into the proliferative dark zone by in vivo antigen delivery to synchronize GC B cells at the time of positive selection. Given that MCD-DLBCLs express high levels of BCL2, we crossed our Btg1Q36H mice to the VavP-Bcl2 model. As compared to VavP-Bcl2, VavP-Bcl2+Btg1 Q36H mice displayed shorter survival (p=0.0005), earlier onset of lymphoma, dysplastic B cell infiltration into non lymphoid organs and they contained highly mutated, selected and clonal tumor B cells. Moribund VavP-Bcl2+Btg1 Q36H mice uniquely featured sheets of large, immunoblastic lymphoma cells, characteristic of ABC-DLBCLs. Most notably, examining ABC-DLBCLs from 5 independent cohorts showed inferior clinical outcome for BTG1 mutant patients (p=0.0011) and independent association of BTG1 mutation with inferior overall survival by multivariable Cox regression (p=0.0190). Collectively, we find that BTG1 mutations mediate lymphomagenesis through an entirely novel mechanism of action that recapitulates the embryonic MYC-dependent "super-competitive" phenotype originally described in Drosophila imaginal disc cells. In the GC, "super-competition" is provided by BTG1 mutation via a subtle acceleration of MYC induction and GC dynamics, conferring dramatic fitness and the potential to transform into aggressive lymphomas. Disclosures Hoehn: Prellis Biologics: Consultancy. Elemento: Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Owkin: Consultancy, Other: Current equity holder; Champions Oncology: Consultancy; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding. Scott: NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; AstraZeneca: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Rich/Genentech: Research Funding. Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees.