RESUMO
The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the mammalian proton-coupled peptide transporter, PepT2, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of antibiotic recognition and the important role of protonation in drug binding and transport.
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Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.
Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/química , Transporte Proteico , Proteínas de Transporte/metabolismoRESUMO
The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Flagelos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Flagelos/metabolismo , Flagelos/química , Flagelos/ultraestrutura , Corpos Basais/metabolismo , Corpos Basais/química , Modelos Moleculares , Rotação , Conformação Proteica , Salmonella/metabolismo , Salmonella/química , Salmonella typhimurium/metabolismo , Salmonella typhimurium/químicaRESUMO
The type VII protein secretion system (T7SS) is found in many Gram-positive bacteria and in pathogenic mycobacteria. All T7SS substrate proteins described to date share a common helical domain architecture at the N-terminus that typically interacts with other helical partner proteins, forming a composite signal sequence for targeting to the T7SS. The C-terminal domains are functionally diverse and in Gram-positive bacteria such as Staphylococcus aureus often specify toxic anti-bacterial activity. Here we describe the first example of a class of T7 substrate, TslA, that has a reverse domain organisation. TslA is widely found across Bacillota including Staphylococcus, Enterococcus and Listeria. We show that the S. aureus TslA N-terminal domain is a phospholipase A with anti-staphylococcal activity that is neutralised by the immunity lipoprotein TilA. Two small helical partner proteins, TlaA1 and TlaA2 are essential for T7-dependent secretion of TslA and at least one of these interacts with the TslA C-terminal domain to form a helical stack. Cryo-EM analysis of purified TslA complexes indicate that they share structural similarity with canonical T7 substrates. Our findings suggest that the T7SS has the capacity to recognise a secretion signal present at either end of a substrate.
Assuntos
Proteínas de Bactérias , Toxinas Biológicas , Proteínas de Bactérias/metabolismo , Staphylococcus aureus/metabolismo , Lipase/metabolismo , Toxinas Biológicas/metabolismo , Transporte BiológicoRESUMO
IMPORTANCE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.
Assuntos
Toxinas Biológicas , Sistemas de Secreção Tipo VII , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Tipo VII/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Regulação Bacteriana da Expressão Gênica , Toxinas Biológicas/metabolismoRESUMO
RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.3 Å) and four holo cryo-electron microscopy structures (overall 3.0-3.5 Å, binding pocket 2.9-3.2 Å). The holo dimers exhibit global motions of helical twisting and bending around the dimer interface. A backbone comparison of the apo and holo states reveals a large structural difference in the P6 extension position. The central strand of the binding pocket, junction 6/3, changes from an 'S'- to a 'U'-shaped conformation to accommodate ligand. Furthermore, the binding pocket can partially form under 1 mM Mg2+ and fully form under 10 mM Mg2+ within the bound-like structure in the absence of ligand. Our results not only demonstrate the stabilizing ligand-induced conformational changes in and around the binding pocket but may also provide further insight into the role of the P6 extension in ligand binding and selectivity.
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NOT1, NOT10, and NOT11 form a conserved module in the CCR4-NOT complex, critical for post-transcriptional regulation in eukaryotes, but how this module contributes to the functions of the CCR4-NOT remains poorly understood. Here, we present cryo-EM structures of human and chicken NOT1:NOT10:NOT11 ternary complexes to sub-3 Å resolution, revealing an evolutionarily conserved, flexible structure. Through biochemical dissection studies, which include the Drosophila orthologs, we show that the module assembly is hierarchical, with NOT11 binding to NOT10, which then organizes it for binding to NOT1. A short proline-rich motif in NOT11 stabilizes the entire module assembly.
Assuntos
Ribonucleases , Fatores de Transcrição , Humanos , Ligação Proteica , Receptores CCR4/metabolismo , Ribonucleases/química , Fatores de Transcrição/metabolismoRESUMO
The type VIIb protein secretion system (T7SSb) plays a role in interbacterial competition in Gram-positive Firmicute bacteria and secretes various toxic effector proteins. The mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins, EsxB, EsxC and EsxD. Here we show that EsxBCD bind to the transport domain of EsaD and function as EsaD export factors. We report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers a conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.
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The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.
Assuntos
Proteína Huntingtina , Proteínas Nucleares , Microscopia Crioeletrônica , Proteína Huntingtina/química , Proteínas Nucleares/química , HumanosRESUMO
Urease is a nickel (Ni) enzyme that is essential for the colonization of Helicobacter pylori in the human stomach. To solve the problem of delivering the toxic Ni ion to the active site without diffusing into the cytoplasm, cells have evolved metal carrier proteins, or metallochaperones, to deliver the toxic ions to specific protein complexes. Ni delivery requires urease to form an activation complex with the urease accessory proteins UreFD and UreG. Here, we determined the cryo-electron microscopy structures of H. pylori UreFD/urease and Klebsiella pneumoniae UreD/urease complexes at 2.3- and 2.7-angstrom resolutions, respectively. Combining structural, mutagenesis, and biochemical studies, we show that the formation of the activation complex opens a 100-angstrom-long tunnel, where the Ni ion is delivered through UreFD to the active site of urease.
Assuntos
Helicobacter pylori , Urease , Humanos , Urease/química , Urease/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Bactérias/metabolismo , Helicobacter pylori/química , Níquel/química , Níquel/metabolismo , KlebsiellaRESUMO
The bacterial flagellum is a macromolecular protein complex that harvests energy from ion-flow across the inner membrane to power bacterial swimming in viscous fluids via rotation of the flagellar filament. Bacteria such as Salmonella enterica are capable of bi-directional flagellar rotation even though ion flow is uni-directional. How uni-directional ion-movement through the inner membrane is utilized by this macromolecular machine to drive bi-directional flagellar rotation is not understood, but a chemotactic response regulator in the cytoplasm is known to reverse the direction of rotation. We here present cryo-EM structures of intact Salmonella flagellar basal bodies, including the cytoplasmic complexes required for power transmission, in conformations representing both directions of rotation. The structures reveal that the conformational changes required for switching the direction of rotation involve 180 degree rotations of both the N- and C-terminal domains of the FliG protein. Combining these models with a new, high-resolution, cryo-EM structure of the MotA5B2 stator, in complex with the C-terminal domain of FliG, reveals how uni-directional ion-flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.
RESUMO
Gliding motility using cell surface adhesins, and export of proteins by the type IX secretion system (T9SS) are two phylum-specific features of the Bacteroidetes. Both of these processes are energized by the GldLM motor complex, which transduces the proton motive force at the inner membrane into mechanical work at the outer membrane. We previously used cryo-electron microscopy to solve the structure of the GldLM motor core from Flavobacterium johnsoniae at 3.9-Å resolution (R. Hennell James, J. C. Deme, A. Kjaer, F. Alcock, et al., Nat Microbiol 6:221-233, 2021, https://dx.doi.org/10.1038/s41564-020-00823-6). Here, we present structures of homologous complexes from a range of pathogenic and environmental Bacteroidetes species at up to 3.0-Å resolution. These structures show that the architecture of the GldLM motor core is conserved across the Bacteroidetes phylum, although there are species-specific differences at the N terminus of GldL. The resolution improvements reveal a cage-like structure that ties together the membrane-proximal cytoplasmic region of GldL and influences gliding function. These findings add detail to our structural understanding of bacterial ion-driven motors that drive the T9SS and gliding motility. IMPORTANCE Many bacteria in the Bacteroidetes phylum use the type IX secretion system to secrete proteins across their outer membrane. Most of these bacteria can also glide across surfaces using adhesin proteins that are propelled across the cell surface. Both secretion and gliding motility are driven by the GldLM protein complex, which forms a nanoscale electrochemical motor. We used cryo-electron microscopy to study the structure of the GldLM protein complex from different species, including the human pathogens Porphyromonas gingivalis and Capnocytophaga canimorsus. The organization of the motor is conserved across species, but we find species-specific structural differences and resolve motor features at higher resolution. This work improves our understanding of the type IX secretion system, which is a virulence determinant in human and animal diseases.
Assuntos
Proteínas de Bactérias , Sistemas de Secreção Bacterianos , Bacteroidetes , Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Bacteroidetes/metabolismo , Microscopia CrioeletrônicaRESUMO
Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.
Assuntos
Proteínas de Artrópodes/química , Proteínas de Artrópodes/imunologia , Inativadores do Complemento/química , Inativadores do Complemento/imunologia , Properdina/imunologia , Rhipicephalus/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Ativação do Complemento , Complemento C3/química , Complemento C3/imunologia , Via Alternativa do Complemento , Humanos , Cinética , Properdina/química , Properdina/genética , Rhipicephalus/química , Rhipicephalus/genética , Alinhamento de SequênciaRESUMO
Shigella sonnei is a major cause of bacillary dysentery and an increasing concern due to the spread of multidrug resistance. S. sonnei harbors pINV, an â¼210 kb plasmid that encodes a type III secretion system (T3SS), which is essential for virulence. During growth in the laboratory, avirulence arises spontaneously in S. sonnei at high frequency, hampering studies on and vaccine development against this important pathogen. Here, we investigated the molecular basis for the emergence of avirulence in S. sonnei and showed that avirulence mainly results from pINV loss, which is consistent with previous findings. Ancestral deletions have led to the loss from S. sonnei pINV of two toxin-antitoxin (TA) systems involved in plasmid maintenance, CcdAB and GmvAT, which are found on pINV in Shigella flexneri. We showed that the introduction of these TA systems into S. sonnei pINV reduced but did not eliminate pINV loss, while the single amino acid polymorphisms found in the S. sonnei VapBC TA system compared with S. flexneri VapBC also contributed to pINV loss. Avirulence also resulted from deletions of T3SS-associated genes in pINV through recombination between insertion sequences (ISs) on the plasmid. These events differed from those observed in S. flexneri due to the different distribution and repertoire of ISs. Our findings demonstrated that TA systems and ISs influenced plasmid dynamics and loss in S. sonnei and could be exploited for the design and evaluation of vaccines. IMPORTANCE Shigella sonnei is the major cause of shigellosis in high-income and industrializing countries and is an emerging, multidrug-resistant pathogen. A significant challenge when studying this bacterium is that it spontaneously becomes avirulent during growth in the laboratory through loss of its virulence plasmid (pINV). Here, we deciphered the mechanisms leading to avirulence in S. sonnei and how the limited repertoire and amino acid sequences of plasmid-encoded toxin-antitoxin (TA) systems make the maintenance of pINV in this bacterium less efficient compared with Shigella flexneri. Our findings highlighted how subtle differences in plasmids in closely related species have marked effects and could be exploited to reduce plasmid loss in S. sonnei. This should facilitate research on this bacterium and vaccine development.
Assuntos
Antitoxinas , Disenteria Bacilar , Sistemas Toxina-Antitoxina , Sequência de Aminoácidos , Antitoxinas/genética , Elementos de DNA Transponíveis , Disenteria Bacilar/microbiologia , Disenteria Bacilar/prevenção & controle , Humanos , Plasmídeos/genética , Shigella flexneri/genética , Shigella sonnei/genética , Sistemas Toxina-Antitoxina/genética , Virulência/genéticaRESUMO
OBJECTIVES: The outbreak of COVID-19 in Massachusetts may have reduced ambulatory care access. Our study aimed to quantify this impact among populations with severely uncontrolled diabetes and hypertension; these populations are at greatest risk for adverse outcomes caused by disruptions in care. METHODS: We analyzed multidisciplinary ambulatory electronic health record data from MDPHnet. We established 3 cohorts of patients with severely uncontrolled diabetes and 3 cohorts of patients with severely uncontrolled hypertension using 2017, 2018, and 2019 data, then followed each cohort through the subsequent 15 months. For the diabetes cohorts, we generated quarterly counts of glycated hemoglobin A1c (HbA1c) tests. For the hypertension cohorts, we generated monthly counts of blood pressure measurements. Finally, we assessed telehealth use among the 2019 diabetes and hypertension cohorts from January 2020 through March 2021. RESULTS: HbA1c testing and blood pressure monitoring dropped considerably during the pandemic compared with previous years. In the 2019 diabetes cohort, HbA1c measurements declined from 44.0% in January-March 2020 (baseline) to 15.9% in April-June 2020 and was 11.8 percentage points below baseline in January-March 2021. In the 2019 hypertension cohort, blood pressure measurements declined from 40.0% in January 2020 to 4.5% in April 2020 and was 23.5 percentage points below baseline in March 2021. Telehealth use increased precipitously during the pandemic but was not uniform across subpopulations. CONCLUSIONS: Access to selected diabetes and hypertension services declined sharply during the pandemic among populations with severely uncontrolled disease. Although telehealth is an important strategy, ensuring equity in access is essential. Telehealth hybrid models can also minimize disruptions in care.
Assuntos
Assistência Ambulatorial/estatística & dados numéricos , COVID-19 , Diabetes Mellitus/prevenção & controle , Acessibilidade aos Serviços de Saúde/estatística & dados numéricos , Hipertensão/prevenção & controle , Adulto , Idoso , Determinação da Pressão Arterial , Estudos de Coortes , Registros Eletrônicos de Saúde/estatística & dados numéricos , Feminino , Hemoglobinas Glicadas , Humanos , Masculino , Massachusetts/epidemiologia , Pessoa de Meia-Idade , Gravidade do Paciente , Telemedicina , Adulto JovemRESUMO
Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc- is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc- is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc- in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.
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Antiporters/química , Antiporters/metabolismo , Cistina/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/biossíntese , Sistema y+ de Transporte de Aminoácidos/química , Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/genética , Bioquímica , Microscopia Crioeletrônica , Cisteína/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/química , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Células HEK293 , Humanos , Neoplasias , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Huntington's disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington's disease and illuminate the structural consequences of HTT polyglutamine expansion.
Assuntos
Éxons , Proteína Huntingtina/genética , Doença de Huntington/genética , Proteínas Nucleares/genética , Peptídeos/metabolismo , Microscopia Crioeletrônica , Humanos , Proteína Huntingtina/metabolismo , Proteína Huntingtina/ultraestrutura , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestruturaRESUMO
Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen.
Assuntos
Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Lipídeos de Membrana , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triglicerídeos/metabolismoRESUMO
Accurate Notch signalling is critical for development and homeostasis. Fine-tuning of Notch-ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N-terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so-called ß1-2 loop that is involved in phospholipid binding. Mutations in the ß1-2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the ß1-2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss-of-function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine-tuning the balance of trans and cis ligand-receptor interactions.