Human members of the solute carrier 1 (SLC1) family of transporters take up excitatory neurotransmitters in the brain and amino acids in peripheral organs. Dysregulation of the function of SLC1 ...transporters is associated with neurodegenerative disorders and cancer. Here we present crystal structures of a thermostabilized human SLC1 transporter, the excitatory amino acid transporter 1 (EAAT1), with and without allosteric and competitive inhibitors bound. The structures reveal architectural features of the human transporters, such as intra- and extracellular domains that have potential roles in transport function, regulation by lipids and post-translational modifications. The coordination of the allosteric inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry reveal a mechanism of inhibition, in which the transporter is locked in the outward-facing states of the transport cycle. Our results provide insights into the molecular mechanisms underlying the function and pharmacology of human SLC1 transporters.
Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. Once the developability of a mAb drug candidate has been assessed, an important step is ...to check its in vivo stability through pharmacokinetics (PK) studies. The gold standard is ligand‐binding assay (LBA) and liquid chromatography‐mass spectrometry (LC‐MS) performed at the peptide level (bottom‐up approach). However, these analytical techniques do not allow to address the different mAb proteoforms that can arise from biotransformation. In recent years, top‐down and middle‐down mass spectrometry approaches have gained popularity to characterize proteins at the proteoform level but are not yet widely used for PK studies. We propose here a workflow based on an automated immunocapture followed by top‐down and middle‐down liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) approaches to characterize mAb proteoforms spiked in mouse plasma. We demonstrate the applicability of our workflow on a large concentration range using pembrolizumab as a model. We also compare the performance of two state‐of‐the‐art Orbitrap platforms (Tribrid Eclipse and Exploris 480) for these studies. The added value of our workflow for an accurate and sensitive characterization of mAb proteoforms in mouse plasma is highlighted.
In antibody‐based drug research, a complete characterization of antibody proteoforms covering both the amino acid sequence and all posttranslational modifications remains a major concern. The usual ...mass spectrometry‐based approach to achieve this goal is bottom‐up proteomics, which relies on the digestion of antibodies but does not allow the diversity of proteoforms to be assessed. Middle‐down and top‐down approaches have recently emerged as attractive alternatives but are not yet mastered and thus used in routine by many analytical chemistry laboratories. The work described here aims at providing guidelines to achieve the best sequence coverage for the fragmentation of intact light and heavy chains generated from a simple reduction of intact antibodies using Orbitrap mass spectrometry. Three parameters were found crucial to this aim: the use of an electron‐based activation technique, the multiplex selection of precursor ions of different charge states, and the combination of replicates.
Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery ...localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.
Bacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, ...comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.
The bacterial type VI secretion system (T6SS) is a macromolecular machine that injects effectors into prokaryotic and eukaryotic cells. The mode of action of the T6SS is similar to contractile ...phages: the contraction of a sheath structure pushes a tube topped by a spike into target cells. Effectors are loaded onto the spike or confined into the tube. In enteroaggregative Escherichia coli, the Tle1 phospholipase binds the C‐terminal extension of the VgrG trimeric spike. Here, we purify the VgrG–Tle1 complex and show that a VgrG trimer binds three Tle1 monomers and inhibits their activity. Using covalent cross‐linking coupled to high‐resolution mass spectrometry, we provide information on the sites of contact and further identify the requirement for a Tle1 N‐terminal secretion sequence in complex formation. Finally, we report the 2.6‐Å‐resolution cryo‐electron microscopy tri‐dimensional structure of the (VgrG)3–(Tle1)3 complex revealing how the effector binds its cargo, and how VgrG inhibits Tle1 phospholipase activity. The inhibition of Tle1 phospholipase activity once bound to VgrG suggests that Tle1 dissociation from VgrG is required upon delivery.
Synopsis
Bacterial Type VI secretion system (T6SS) effectors are loaded in the T6SS tube or on the spike complex for delivery into target cells. The current study presents the detailed structure of the T6SS effector Tle1 in complex with the VgrG spike of enteroaggregative Escherichia coli.
VgrG trimer binds three Tle1 monomers.
VgrG inhibits Tle1 phospholipase activity.
N‐terminal secretion sequence of Tle1 interacts with the VgrG C‐terminal TTR domain.
The 2.6‐Å resolution cryo‐EM structure of the (VgrG)3‐(Tle1)3 complex reveals a triskelion‐like assembly.
A cryo‐EM structure reveals interactions between the Type VI secretion system effector Tle1 with the spike protein VgrG from enteroaggregative Escherichia coli.
Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade ...type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.
Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and ...co‐transport transmitter into the cytoplasm with Na+ and H+, while counter‐transporting K+ to re‐initiate the transport cycle. However, the molecular mechanisms underlying ion‐coupled transport remain incompletely understood. Here, we present 3D X‐ray crystallographic and cryo‐EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter‐transport ion bound states. Binding energies of Na+ and H+, and unexpectedly Ca2+, are coupled to neurotransmitter binding. Ca2+ competes for a conserved Na+ site, suggesting a regulatory role for Ca2+ in glutamate transport at the synapse, while H+ binds to a conserved glutamate residue stabilizing substrate occlusion. The counter‐transported ion binding site overlaps with that of glutamate, revealing the K+‐based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
Synopsis
The mechanism by which excitatory amino acid transporters (EAATs) in the brain use ionic transmembrane gradients to ensure glutamate uptake from the synaptic cleft remains incompletely understood. This work combines structural data and thermodynamic analyses to describe a complete transport cycle of human EAAT1 in different ion bound conformations.
EAAT1 Na+‐coupling is conserved from archaea to humans.
EAAT1 H+‐coupling involves a conserved glutamate residue in the transport domain.
Binding sites for glutamate and the counter‐transported K+ overlap.
Ca2+ competes for a conserved Na+ site and is thermodynamically coupled to neurotransmitter binding.
Comprehensive structural and thermodynamic analyses of the synaptic glutamate transporter EAAT1 highlights overlapping binding sites and unexpected competition by Ca2+.
Type IV pili (TFP) are multifunctional micrometer‐long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of ...the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto‐aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto‐aggregation, pili must reach a minimum length to allow pilus–pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip‐located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP‐dependent aggregation and adhesion.
Synopsis
Adhesion mediated by type IV pili enables host invasion in a range of mainly Gram‐negative bacterial pathogens. Deep mutational scanning of the Neisseria meningitidis major pilin PilE uncovers key steps of the type IV pili‐dependent adhesion process.
Interaction via the pilus tip initiates bacterial adhesion to the cell surface.
During the later phase of host cell adhesion, residues along the length of the pili bind to the cell surface.
A threshold pilus length is required to initiate pilus‐pilus interactions leading to bacterial auto‐aggregation and formation of bacterial microcolonies.
Systematic mutations in pilE uncover two successive modes of type IV pilus‐mediated host cell adhesion and the requirement for a pilus length threshold for bacterial aggregation in the meningococcus.
Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic trimers. The rod‐like E subunits bend ...into “hairpins” to bring the fusion loops next to the C‐terminal transmembrane (TM) anchors, with the TM‐proximal “stem” element zippering the E trimer to force apposition of the membranes. The structure of the complete class II trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E‐protein trimers from the tick‐borne encephalitis flavivirus with sequential stem truncations. Our thermostability and antibody‐binding data suggest that the stem “zipper” ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to trimer stability. We further identified a highly dynamic behavior of E trimers C‐terminally truncated upstream the CS, which, unlike fully stem‐zippered trimers, undergo rapid deuterium exchange at the trimer interface. These results thus identify important “breathing” intermediates in the E‐protein‐driven membrane fusion process.
Synopsis
The flavivirus envelope protein E exhibits a dynamic “breathing” behavior not only as a dimer at the surface of mature virions, but also as a trimeric intermediate during membrane fusion in the endosome.
The stem element of the flavivirus protein E is important as “zipper” during the formation of its final post‐fusion structure.
The stem zipper ends at a characteristic conserved sequence that cloaks the fusion loops at the tip of protein E.
Deuterium exchange experiments identify “breathing” intermediates during protein E‐driven membrane fusion.
The flavivirus envelope protein E exhibits a dynamic “breathing” behavior not only as a dimer at the surface of mature virions, but also as a trimeric intermediate during membrane fusion in the endosome.
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