Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc.

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.

Structural properties of the HNF-1A transactivation domain.

Hepatocyte nuclear factor 1α (HNF-1A) is a transcription factor with important gene regulatory roles in pancreatic ß-cells. HNF1A gene variants are associated with a monogenic form of diabetes (HNF1A-MODY) or an increased risk for type 2 diabetes. While several pancreatic target genes of HNF-1A have been described, a lack of knowledge regarding the structure-function relationships in HNF-1A prohibits a detailed understanding of HNF-1A-mediated gene transcription, which is important for precision medicine and improved patient care. Therefore, we aimed to characterize the understudied transactivation domain (TAD) of HNF-1A in vitro. We present a bioinformatic approach to dissect the TAD sequence, analyzing protein structure, sequence composition, sequence conservation, and the existence of protein interaction motifs. Moreover, we developed the first protocol for the recombinant expression and purification of the HNF-1A TAD. Small-angle X-ray scattering and synchrotron radiation circular dichroism suggested a disordered conformation for the TAD. Furthermore, we present functional data on HNF-1A undergoing liquid-liquid phase separation, which is in line with in silico predictions and may be of biological relevance for gene transcriptional processes in pancreatic ß-cells.

Conformational analysis of membrane-proximal segments of GDAP1 in a lipidic environment using synchrotron radiation suggests a mode of assembly at the mitochondrial outer membrane.

The mitochondrial outer membrane creates a diffusion barrier between the cytosol and the mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflets of the mitochondrial outer membrane in different orientations. Impairment of these interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Identification of structural determinants of nicotinamide phosphoribosyl transferase (NAMPT) activity and substrate selectivity.

NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD+ by sirtuins, PARPs, and other NAD+-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

The intrinsically disordered protein glue of the myelin major dense line: Linking AlphaFold2 predictions to experimental data.

Numerous human proteins are classified as intrinsically disordered proteins (IDPs). Due to their physicochemical properties, high-resolution structural information about IDPs is generally lacking. On the other hand, IDPs are known to adopt local ordered structures upon interactions with e.g. other proteins or lipid membrane surfaces. While recent developments in protein structure prediction have been revolutionary, their impact on IDP research at high resolution remains limited. We took a specific example of two myelin-specific IDPs, the myelin basic protein (MBP) and the cytoplasmic domain of myelin protein zero (P0ct). Both of these IDPs are crucial for normal nervous system development and function, and while they are disordered in solution, upon membrane binding, they partially fold into helices, being embedded into the lipid membrane. We carried out AlphaFold2 predictions of both proteins and analysed the models in light of experimental data related to protein structure and molecular interactions. We observe that the predicted models have helical segments that closely correspond to the membrane-binding sites on both proteins. We furthermore analyse the fits of the models to synchrotron-based X-ray scattering and circular dichroism data from the same IDPs. The models are likely to represent the membrane-bound state of both MBP and P0ct, rather than the conformation in solution. Artificial intelligence-based models of IDPs appear to provide information on the ligand-bound state of these proteins, instead of the conformers dominating free in solution. We further discuss the implications of the predictions for mammalian nervous system myelination and their relevance to understanding disease aspects of these IDPs.

Conserved intramolecular networks in GDAP1 are closely connected to CMT-linked mutations and protein stability.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices ⍺3, ⍺7, and ⍺8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. GDAP1-like proteins branched early from the larger group of GSTs. Phylogenetic calculations could not resolve the exact early chronology, but the evolution of GDAP1 is roughly as old as the splits of archaea from other kingdoms. Many known CMT mutation sites involve conserved residues or interact with them. A central role for the ⍺6-⍺7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration.

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase.

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0-1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

Exome sequencing in a child with neurodevelopmental disorder and epilepsy: Variant analysis of the AHNAK2 gene.

BACKGROUND: The AHNAK2 gene encodes a large nucleoprotein expressed in several tissues, including brain, squamous epithelia, smooth muscle, and neuropil. Its role in calcium signaling has been suggested and to date, clear evidence about its involvement in the pathogenesis of clinical disorders is still lacking. METHODS: Here, we report a female 24-year-old patient diagnosed with a cardio-facio-cutaneous-like phenotype (CFC-like), characterized by epilepsy, psychomotor development delay, atopic dermatitis, congenital heart disease, hypotonia, and facial dysmorphism, who is compound heterozygote for two missense mutations in the AHNAK2 gene detected by exome sequencing. RESULTS: This patient had no detectable variant in any of the genes known to be associated with the cardio-facio-cutaneous syndrome. Moreover, the mode of inheritance does not appear to be autosomal dominant, as it is in typical CFC syndrome. We have performed in silico assessment of mutation severity separately for each missense mutation, but this analysis excludes a severe effect on protein function. Protein structure predictions indicate the mutations are located in flexible regions possibly involved in molecular interactions. CONCLUSION: We discuss an alternative interpretation on the potential involvement of the two missense mutations in the AHNAK2 gene on the expression of CFC-like phenotype in this patient based on inter-allelic complementation.

Human myelin proteolipid protein structure and lipid bilayer stacking.

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure-function differences, as well as fundamental protein-lipid interplay in CNS compact myelin.

High-affinity anti-Arc nanobodies provide tools for structural and functional studies.

Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, we produced and characterised six high-affinity anti-Arc nanobodies (Nb). The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that we have trapped a capsid-like conformation of the human Arc CTD. Our data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity.

Structural insights into Charcot-Marie-Tooth disease-linked mutations in human GDAP1.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its different subtypes are linked to mutations in dozens of different genes. Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause two types of CMT, demyelinating CMT4A and axonal CMT2K. The GDAP1-linked CMT genotypes are mainly missense point mutations. Despite clinical profiling and in vivo studies on the mutations, the etiology of GDAP1-linked CMT is poorly understood. Here, we describe the biochemical and structural properties of the Finnish founding CMT2K mutation H123R and CMT2K-linked R120W, both of which are autosomal dominant mutations. The disease variant proteins retain close to normal structure and solution behavior, but both present a significant decrease in thermal stability. Using GDAP1 variant crystal structures, we identify a side-chain interaction network between helices ⍺3, ⍺6, and ⍺7, which is affected by CMT mutations, as well as a hinge in the long helix ⍺6, which is linked to structural flexibility. Structural analysis of GDAP1 indicates that CMT may arise from disruption of specific intra- and intermolecular interaction networks, leading to alterations in GDAP1 structure and stability, and, eventually, insufficient motor and sensory neuron function.

Erratum: Structure of the Complete Dimeric Human GDAP1 Core Domain Provides Insights Into Ligand Binding and Clustering of Disease Mutations.

[This corrects the article DOI: 10.3389/fmolb.2020.631232.].

Development and Validation of Arc Nanobodies: New Tools for Probing Arc Dynamics and Function.

Activity-regulated cytoskeleton-associated (Arc) protein plays key roles in long-term synaptic plasticity, memory, and cognitive flexibility. However, an integral understanding of Arc mechanisms is lacking. Arc is proposed to function as an interaction hub in neuronal dendrites and the nucleus, yet Arc can also form retrovirus-like capsids with proposed roles in intercellular communication. Here, we sought to develop anti-Arc nanobodies (ArcNbs) as new tools for probing Arc dynamics and function. Six ArcNbs representing different clonal lines were selected from immunized alpaca. Immunoblotting with recombinant ArcNbs fused to a small ALFA-epitope tag demonstrated binding to recombinant Arc as well as endogenous Arc from rat cortical tissue. ALFA-tagged ArcNb also provided efficient immunoprecipitation of stimulus-induced Arc after carbachol-treatment of SH-SY5Y neuroblastoma cells and induction of long-term potentiation in the rat dentate gyrus in vivo. Epitope mapping showed that all Nbs recognize the Arc C-terminal region containing the retroviral Gag capsid homology domain, comprised of tandem N- and C-lobes. ArcNbs E5 and H11 selectively bound the N-lobe, which harbors a peptide ligand binding pocket specific to mammals. Four additional ArcNbs bound the region containing the C-lobe and C-terminal tail. For use as genetically encoded fluorescent intrabodies, we show that ArcNbs fused to mScarlet-I are uniformly expressed, without aggregation, in the cytoplasm and nucleus of HEK293FT cells. Finally, mScarlet-I-ArcNb H11 expressed as intrabody selectively bound the N-lobe and enabled co-immunoprecipitation of full-length intracellular Arc. ArcNbs are versatile tools for live-cell labeling and purification of Arc, and interrogation of Arc capsid domain specific functions.

Structural and biophysical characterization of transcription factor HNF-1A as a tool to study MODY3 diabetes variants.

Hepatocyte nuclear factor 1A (HNF-1A) is a transcription factor expressed in several embryonic and adult tissues, modulating the expression of numerous target genes. Pathogenic variants in the HNF1A gene are known to cause maturity-onset diabetes of the young 3 (MODY3 or HNF1A MODY), a disease characterized by dominant inheritance, age of onset before 25 to 35 years of age, and pancreatic ß-cell dysfunction. A precise diagnosis can alter management of this disease, as insulin can be exchanged with sulfonylurea tablets and genetic counseling differs from polygenic forms of diabetes. Therefore, more knowledge on the mechanisms of HNF-1A function and the level of pathogenicity of the numerous HNF1A variants is required for precise diagnostics. Here, we structurally and biophysically characterized an HNF-1A protein containing both the DNA-binding domain and the dimerization domain, and determined the folding and DNA-binding capacity of two established MODY3 HNF-1A variant proteins (P112L, R263C) and one variant of unknown significance (N266S). All three variants showed reduced functionality compared to the WT protein. Furthermore, while the R263C and N266S variants displayed reduced binding to an HNF-1A target promoter, we found the P112L variant was unstable in vitro and in cells. Our results support and mechanistically explain disease causality for these investigated variants and present a novel approach for the dissection of structurally unstable and DNA-binding defective variants. This study indicates that structural and biochemical investigation of HNF-1A is a valuable tool in reliable variant classification needed for precision diabetes diagnostics and management.

Structure and function of an atypical homodimeric actin capping protein from the malaria parasite.

Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the ß-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a ß-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.

Correction: Raasakka, A.; Kursula, P. Flexible Players within the Sheaths: The Intrinsically Disordered Proteins of Myelin in Health and Disease. Cells 2020, 9, 470.

The authors would like to make corrections to their review [...].

Multiple sclerosis and myelin basic protein: insights into protein disorder and disease.

Myelin basic protein (MBP) is an abundant protein in central nervous system (CNS) myelin. MBP has long been studied as a factor in the pathogenesis of the autoimmune neurodegenerative disease multiple sclerosis (MS). MS is characterized by CNS inflammation, demyelination, and axonal loss. One of the main theories on the pathogenesis of MS suggests that exposure to foreign antigens causes the activation of cross-reactive T cells in genetically susceptible individuals, with MBP being a possible autoantigen. While a direct role for MBP as a primary antigen in human MS is unclear, it is clear that MBP and its functions in myelin formation and long-term maintenance are linked to MS. This review looks at some key molecular characteristics of MBP and its relevance to MS, as well as the mechanisms of possible molecular mimicry between MBP and some viral antigens. We also discuss the use of serum anti-myelin antibodies as biomarkers for disease. MBP is a prime example of an apparently simple, but in fact biochemically and structurally complex molecule, which is closely linked to both normal nervous system development and neurodegenerative disease.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.