Chemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we developed an advanced in vivo cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 3300 cross-links for the LC-MS/MS analysis of a biological triplicate using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein−protein interactions. All raw data, databases, and processing parameters are available on ProteomeXchange via PRIDE repository (data set identifier PXD021553).

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 Ca 2+ , are coupled to neurotransmitter binding. Ca 2+ competes for a conserved Na + site, suggesting a regulatory role for Ca 2+ 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.

Motivation We present a new software-tool allowing an easy visualization of fragment ions and thus a rapid evaluation of key experimental parameters on the sequence coverage obtained for the MS/MS (tandem mass spectrometry) analysis of intact proteins. Our tool can process data obtained from various deconvolution and fragment assignment software. Results We demonstrate that TDFragMapper can rapidly highlight the experimental fragmentation parameters that are critical to the characterization of intact proteins of various size using top-down proteomics. Availability and implementation TDFragMapper, a demonstration video and user tutorial are freely available for academic use at https://msbio.pasteur.fr/tdfragmapper; all data are thus available from the ProteomeXchange consortium (identifier PXD024643). Supplementary information Supplementary data are available at Bioinformatics online.