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 their functions is associated to neurodegenerative disorders and cancer. Here we present the first 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 show novel architectural features of the human transporters, including intra- and extracellular domains with potential roles in transport function, as well as regulation by lipids and post-translational modifications. The coordination of the inhibitor in the structures and the change in the transporter dynamics measured by hydrogen-deuterium exchange mass spectrometry, reveal an allosteric mechanism of inhibition, whereby the transporter is locked in the outward-facing states of the transport cycle. Our results provide unprecedented insights into the molecular mechanisms of function and pharmacology of human SLC1 transporters.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

The Gram-negative bacterium Neisseria meningitidis asymptomatically colonizes the throat of 10 to 30% of the human population, but throat colonization can also act as the port of entry to the blood (septicemia) and then the brain (meningitis). Colonization is mediated by filamentous organelles referred to as type IV pili, which allow the formation of bacterial aggregates associated with host cells. We found that proliferation of N. meningitidis in contact with host cells increased the transcription of a bacterial gene encoding a transferase that adds phosphoglycerol onto type IV pili. This unusual posttranslational modification specifically released type IV pili-dependent contacts between bacteria. In turn, this regulated detachment process allowed propagation of the bacterium to new colonization sites and also migration across the epithelium, a prerequisite for dissemination and invasive disease.

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.

Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. Molecular & Cellular

No abstract

Peptide fragmentation can lead to an oxazolone or diketopiperazine b2+ ion structure. IRMPD spectroscopy combined with computational modeling and gas-phase H/D exchange was used to study the structure of the b2+ ion from protonated HAAAA. The experimental spectrum of the b2+ ion matches both the experimental spectrum for the protonated cyclic dipeptide HA (a commercial diketopiperazine) and the theoretical spectrum for a diketopiperazine protonated at the imidazole pi nitrogen. A characteristic band at 1875 cm−1, and increased abundance of the peaks at 1619 and 1683 cm−1, indicate a second population corresponding to an oxazolone species. H/D exchange also shows a mixture of two populations consistent with a mixture of b2+ ion diketopiperazine and oxazolone structures.

Electron-transfer and -capture dissociations of doubly protonated peptides gave dramatically different product ions for a series of histidine-containing pentapeptides of both non-tryptic (AAHAL, AHAAL, AHADL, AHDAL) and tryptic (AAAHK, AAHAK, AHAAK, HAAAK, AAAHR, AAHAR, AHAAR, HAAAR) type. Electron transfer from gaseous Cs atoms and fluoranthene anions triggered backbone dissociations of all four N-C(alpha) bonds in the peptide ions in addition to loss of H and NH(3). Substantial fractions of charge-reduced cation-radicals did not dissociate on an experimental time scale ranging from 10(-6) to 10(-1) s. Multistage tandem mass spectrometric (MS(n)) experiments indicated that the non-dissociating cation-radicals had undergone rearrangements. These were explained as being due to proton migrations from N-terminal ammonium and COOH groups to the C-2' position of the reduced His ring, resulting in substantial radical stabilization. Ab initio calculations revealed that the charge-reduced cation-radicals can exist as low-energy zwitterionic amide pi* states which were local energy minima. These states underwent facile exothermic proton migrations to form aminoketyl radical intermediates, whereas direct N-C(alpha) bond cleavage in zwitterions was disfavored. RRKM analysis indicated that backbone N-C(alpha) bond cleavages did not occur competitively from a single charge-reduced precursor. Rather, these bond cleavages proceeded from distinct intermediates which originated from different electronic states accessed by electron transfer. In stark contrast to electron transfer, capture of a free electron by the peptide ions mainly induced radical dissociations of the charge-carrying side chains and loss of a hydrogen atom followed by standard backbone dissociations of even-electron ions. The differences in dissociation are explained by different electronic states being accessed upon electron transfer and capture.