Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Assessment of Safety of a Fully Implanted Endovascular Brain-Computer Interface for Severe Paralysis in 4 Patients: The Stentrode With Thought-Controlled Digital Switch (SWITCH) Study.

Importance: Brain-computer interface (BCI) implants have previously required craniotomy to deliver penetrating or surface electrodes to the brain. Whether a minimally invasive endovascular technique to deliver recording electrodes through the jugular vein to superior sagittal sinus is safe and feasible is unknown. Objective: To assess the safety of an endovascular BCI and feasibility of using the system to control a computer by thought. Design, Setting, and Participants: The Stentrode With Thought-Controlled Digital Switch (SWITCH) study, a single-center, prospective, first in-human study, evaluated 5 patients with severe bilateral upper-limb paralysis, with a follow-up of 12 months. From a referred sample, 4 patients with amyotrophic lateral sclerosis and 1 with primary lateral sclerosis met inclusion criteria and were enrolled in the study. Surgical procedures and follow-up visits were performed at the Royal Melbourne Hospital, Parkville, Australia. Training sessions were performed at patients' homes and at a university clinic. The study start date was May 27, 2019, and final follow-up was completed January 9, 2022. Interventions: Recording devices were delivered via catheter and connected to subcutaneous electronic units. Devices communicated wirelessly to an external device for personal computer control. Main Outcomes and Measures: The primary safety end point was device-related serious adverse events resulting in death or permanent increased disability. Secondary end points were blood vessel occlusion and device migration. Exploratory end points were signal fidelity and stability over 12 months, number of distinct commands created by neuronal activity, and use of system for digital device control. Results: Of 4 patients included in analyses, all were male, and the mean (SD) age was 61 (17) years. Patients with preserved motor cortex activity and suitable venous anatomy were implanted. Each completed 12-month follow-up with no serious adverse events and no vessel occlusion or device migration. Mean (SD) signal bandwidth was 233 (16) Hz and was stable throughout study in all 4 patients (SD range across all sessions, 7-32 Hz). At least 5 attempted movement types were decoded offline, and each patient successfully controlled a computer with the BCI. Conclusions and Relevance: Endovascular access to the sensorimotor cortex is an alternative to placing BCI electrodes in or on the dura by open-brain surgery. These final safety and feasibility data from the first in-human SWITCH study indicate that it is possible to record neural signals from a blood vessel. The favorable safety profile could promote wider and more rapid translation of BCI to people with paralysis. Trial Registration: ClinicalTrials.gov Identifier: NCT03834857.

Plasma Neurofilament Light Chain and Clinical Diagnosis in Frontotemporal Dementia Syndromes.

BACKGROUND: Frontotemporal dementia (FTD) syndromes, mimics, phenocopy (phFTD), and slowly progressive behavioral variant FTD (bvFTD) can be difficult to distinguish clinically. Biomarkers such as neurofilament light chain (NfL) may be helpful. OBJECTIVE: To study plasma NfL levels in people with FTD syndromes and determine if plasma NfL can distinguish between FTD syndromes and phFTD. METHODS: Plasma NfL levels were estimated using both Simoa® Quanterix HD-X™ and SR-X™ machines grouped via final diagnosis after investigation and review. RESULTS: Fifty participants were studied: bvFTD = 20, semantic variant FTD (svFTD) = 11, non-fluent variant FTD (nfvFTD) = 9, FTD with motor neuron disease (MND) = 4, phFTD = 2, slow progressors = 3, FTD mimic = 1, mean age 67.2 (SD 8.4) years. NfL levels were significantly higher in the FTD group compared to phenocopy group (p = 0.003). Median NfL (IQR) pg/mL was comparable in the FTD syndromes: bvFTD 41.10 (50.72), svFTD 44.38 (16.61), and nfvFTD 42.61 (22.93), highest in FTD with MND 79.67 (45.32) and lowest in both phFTD 13.99 (0.79) and slow progressors 17.97 (3.62). CONCLUSION: Plasma NfL appears to differentiate FTD syndromes and mimics. However, a lower NfL may predict a slower, but not necessarily absence of neurodegeneration, and therefore appears limited in distinguishing slow progressors from FTD phenocopies. Larger numbers of patients from all clinical groups are required to strengthen diagnostic utility.

Insights on the Quest for the Structure-Function Relationship of the Mitochondrial Pyruvate Carrier.

The molecular identity of the mitochondrial pyruvate carrier (MPC) was presented in 2012, forty years after the active transport of cytosolic pyruvate into the mitochondrial matrix was first demonstrated. An impressive amount of in vivo and in vitro studies has since revealed an unexpected interplay between one, two, or even three protein subunits defining different functional MPC assemblies in a metabolic-specific context. These have clear implications in cell homeostasis and disease, and on the development of future therapies. Despite intensive efforts by different research groups using state-of-the-art computational tools and experimental techniques, MPCs' structure-based mechanism remains elusive. Here, we review the current state of knowledge concerning MPCs' molecular structures by examining both earlier and recent studies and presenting novel data to identify the regulatory, structural, and core transport activities to each of the known MPC subunits. We also discuss the potential application of cryogenic electron microscopy (cryo-EM) studies of MPC reconstituted into nanodiscs of synthetic copolymers for solving human MPC2.

Nano-encapsulated Escherichia coli Divisome Anchor ZipA, and in Complex with FtsZ.

The E. coli membrane protein ZipA, binds to the tubulin homologue FtsZ, in the early stage of cell division. We isolated ZipA in a Styrene Maleic Acid lipid particle (SMALP) preserving its position and integrity with native E. coli membrane lipids. Direct binding of ZipA to FtsZ is demonstrated, including FtsZ fibre bundles decorated with ZipA. Using Cryo-Electron Microscopy, small-angle X-ray and neutron scattering, we determine the encapsulated-ZipA structure in isolation, and in complex with FtsZ to a resolution of 1.6 nm. Three regions can be identified from the structure which correspond to, SMALP encapsulated membrane and ZipA transmembrane helix, a separate short compact tether, and ZipA globular head which binds FtsZ. The complex extends 12 nm from the membrane in a compact structure, supported by mesoscale modelling techniques, measuring the movement and stiffness of the regions within ZipA provides molecular scale analysis and visualisation of the early divisome.

SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis.

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.

Analysis of SMALP co-extracted phospholipids shows distinct membrane environments for three classes of bacterial membrane protein.

Biological characterisation of membrane proteins lags behind that of soluble proteins. This reflects issues with the traditional use of detergents for extraction, as the surrounding lipids are generally lost, with adverse structural and functional consequences. In contrast, styrene maleic acid (SMA) copolymers offer a detergent-free method for biological membrane solubilisation to produce SMA-lipid particles (SMALPs) containing membrane proteins together with their surrounding lipid environment. We report the development of a reverse-phase LC-MS/MS method for bacterial phospholipids and the first comparison of the profiles of SMALP co-extracted phospholipids from three exemplar bacterial membrane proteins with different topographies: FtsA (associated membrane protein), ZipA (single transmembrane helix), and PgpB (integral membrane protein). The data showed that while SMA treatment per se did not preferentially extract specific phospholipids from the membrane, SMALP-extracted ZipA showed an enrichment in phosphatidylethanolamines and depletion in cardiolipins compared to the bulk membrane lipid. Comparison of the phospholipid profiles of the 3 SMALP-extracted proteins revealed distinct lipid compositions for each protein: ZipA and PgpB were similar, but in FtsA samples longer chain phosphatidylglycerols and phosphatidylethanolamines were more abundant. This method offers novel information on the phospholipid interactions of these membrane proteins.

Membrane protein nanoparticles: the shape of things to come.

The use of styrene-maleic acid (SMA) for the purification of a wide range of membrane proteins (MPs) from both prokaryotic and eukaryotic sources has begun to make an impact in the field of MP biology. This method is growing in popularity as a means to purify and thoroughly investigate the structure and function of MPs and biological membranes. The amphiphilic SMA copolymer can effectively extract MPs directly from a native lipid bilayer to form discs ∼10 nm in diameter. The resulting lipid particles, or styrene-maleic acid lipid particles (SMALPs), contain SMA, protein and membrane lipid. MPs purified in SMALPs are able to retain their native structure and, in many cases, functional activity, and growing evidence suggests that MPs purified using SMA have enhanced thermal stability compared with detergent-purified proteins. The SMALP method is versatile and is compatible with a wide range of cell types across taxonomic domains. It can readily be adapted to replace detergent in many protein purification methods, often with only minor changes made to the existing protocol. Moreover, biophysical analysis and structural determination may now be a possibility for many large, unstable MPs. Here, we review recent advances in the area of SMALP purification and how it is affecting the field of MP biology, critically assess recent progress made with this method, address some of the associated technical challenges which may remain unresolved and discuss opportunities for exploiting SMALPs to expand our understanding of structural and functional properties of MPs.

Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer.

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Membrane proteins: is the future disc shaped?

The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology.

Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC.

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein 'insertase' YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) - a supercomplex of SecYEG-SecDF-YajC-YidC - is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

A method for detergent-free isolation of membrane proteins in their local lipid environment.

Despite the great importance of membrane proteins, structural and functional studies of these proteins present major challenges. A significant hurdle is the extraction of the functional protein from its natural lipid membrane. Traditionally achieved with detergents, purification procedures can be costly and time consuming. A critical flaw with detergent approaches is the removal of the protein from the native lipid environment required to maintain functionally stable protein. This protocol describes the preparation of styrene maleic acid (SMA) co-polymer to extract membrane proteins from prokaryotic and eukaryotic expression systems. Successful isolation of membrane proteins into SMA lipid particles (SMALPs) allows the proteins to remain with native lipid, surrounded by SMA. We detail procedures for obtaining 25 g of SMA (4 d); explain the preparation of protein-containing SMALPs using membranes isolated from Escherichia coli (2 d) and control protein-free SMALPS using E. coli polar lipid extract (1-2 h); investigate SMALP protein purity by SDS-PAGE analysis and estimate protein concentration (4 h); and detail biophysical methods such as circular dichroism (CD) spectroscopy and sedimentation velocity analytical ultracentrifugation (svAUC) to undertake initial structural studies to characterize SMALPs (∼2 d). Together, these methods provide a practical tool kit for those wanting to use SMALPs to study membrane proteins.

Encapsulated membrane proteins: A simplified system for molecular simulation.

Over the past 50years there has been considerable progress in our understanding of biomolecular interactions at an atomic level. This in turn has allowed molecular simulation methods employing full atomistic modelling at ever larger scales to develop. However, some challenging areas still remain where there is either a lack of atomic resolution structures or where the simulation system is inherently complex. An area where both challenges are present is that of membranes containing membrane proteins. In this review we analyse a new practical approach to membrane protein study that offers a potential new route to high resolution structures and the possibility to simplify simulations. These new approaches collectively recognise that preservation of the interaction between the membrane protein and the lipid bilayer is often essential to maintain structure and function. The new methods preserve these interactions by producing nano-scale disc shaped particles that include bilayer and the chosen protein. Currently two approaches lead in this area: the MSP system that relies on peptides to stabilise the discs, and SMALPs where an amphipathic styrene maleic acid copolymer is used. Both methods greatly enable protein production and hence have the potential to accelerate atomic resolution structure determination as well as providing a simplified format for simulations of membrane protein dynamics. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy.

Despite the great progress recently made in resolving their structures, investigation of the structural biology of membrane proteins still presents major challenges. Even with new technical advances such as lipidic cubic phase crystallisation, obtaining well-ordered crystals remains a significant hurdle in membrane protein X-ray crystallographic studies. As an alternative, electron microscopy has been shown to be capable of resolving >3.5Å resolution detail in membrane proteins of modest (~300 kDa) size, without the need for crystals. However, the conventional use of detergents for either approach presents several issues, including the possible effects on structure of removing the proteins from their natural membrane environment. As an alternative, it has recently been demonstrated that membrane proteins can be effectively isolated, in the absence of detergents, using a styrene maleic acid co-polymer (SMA). This approach yields SMA lipid particles (SMALPs) in which the membrane proteins are surrounded by a small disk of lipid bilayer encircled by polymer. Here we use the Escherichia coli secondary transporter AcrB as a model membrane protein to demonstrate how a SMALP scaffold can be used to visualise membrane proteins, embedded in a near-native lipid environment, by negative stain electron microscopy, yielding structures at a modest resolution in a short (days) timeframe. Moreover, we show that AcrB within a SMALP scaffold is significantly more active than the equivalent DDM stabilised form. The advantages of SMALP scaffolds within electron microscopy are discussed and we conclude that they may prove to be an important tool in studying membrane protein structure and function.

Cerebrospinal fluid pressure in adults.

BACKGROUND: Lumbar puncture (LP) is a widely-used investigative procedure. It allows relatively non-invasive measurement of intracranial pressure (ICP) which may have a significant impact on diagnosis and/or patient management. Over the years there has been considerable discussion about various aspects of the procedure, including what constitutes a normal opening pressure, what factors might influence this, and how LP is best performed. EVIDENCE ACQUISITION: A review of the literature was carried out by searching PubMed and Medline, scanning relevant medical journals for recent publications, and carrying out secondary referencing and contacting other clinicians, where appropriate. RESULTS: The normal range of ICP measured by LP in adults in a typical clinical setting should now be regarded as 6 to 25 cmH2O (95% confidence intervals), with a population mean of about 18 cmH2O. There is, however, considerable variability: some normal individuals have pressures of 30 cmH2O (or, occasionally, even higher) meaning that pressure measurements must be interpreted in the clinical context. CONCLUSIONS: This article aims to provide the practicing neuro-ophthalmologist with up-to-date information about the ways in which various factors can influence pressure measurements obtained at LP.

Propagule supply controls grazer community structure and primary production in a benthic marine ecosystem.

Early theories of species diversity proposed that communities at equilibrium are saturated with species. However, experiments in plant communities suggest that many communities are unsaturated and species richness can be increased by adding propagules of new species. We experimentally tested for community saturation and measured the effects of propagule supply on community structure in a benthic marine system. We manipulated propagule supply (arrival of individuals of numerous species) of mobile grazers in experimental mesocosms over multiple generations and, unlike previous tests, we examined the cascading effects of propagule supply on prey (macroalgae) biomass. We found little evidence for saturation, despite the absence of processes such as disturbance and predation that are thought to alleviate saturation in nature. Increasing propagule supply increased the total number of species and made rare species more abundant. Perhaps surprisingly, given the strong effect of propagule supply on species richness, supply-related changes in body size and composition suggest that competitive interactions remained important. Grazer supply also had strong cascading effects on primary production, possibly because of dietary complementarity modified by territorial behavior. Our results indicate that propagule supply can directly influence the diversity and composition of communities of mobile animals. Furthermore, the supply of consumer propagules can have strong indirect effects on prey and fundamental ecosystem properties.

Macroalgae has no effect on the severity and dynamics of Caribbean yellow band disease.

By removing herbivores and promoting increases in macroalgae, overfishing is thought to indirectly cause coral disease and mortality. We performed three field manipulations to test the general hypothesis that overfishing and the subsequent alteration of coral reef trophic dynamics are a cause of coral epizootics. Specifically, we asked whether the presence of macroalgae can influence within- and among-colony spread rates of Caribbean Yellow Band Disease in Montastraea faveolata. Macroalgae were placed next to infected and healthy, adult and small coral colonies to measure effects on disease spread rate, coral growth and coral survival. Surprisingly, the addition of macroalgae did not affect disease severity or coral fitness. Our results indicate that macroalgae have no effect on the severity and dynamics of Caribbean Yellow Band Disease and that fisheries management alone will not mitigate the effects of this important epizootic.