Project description:we used hydrogen deuterium exchange masss spec (HDX-MS) to define the exchange kinetics of PLC-y1 as it interacts with the kinase domain of FGFR1, and liposomes containing PIP2, in order to understand how PLC-y isozymes operate at membranes. We show that the binding of FGFR1k to PLC-y1 increases deuterium incorporation within the interface between the regulatory domains and the catalytic core. Importantly, the addition of liposomes further increase deuterium exchange within the same region.
Project description:we used HDXMS to study if PI4KA(530-560) is able to bind calcineurin. Here we showed a significant protection in deuterium exchange upon binding calcineurin in the wild type state, indicating these proteins are interacting. Using a mutant that should inhibit the interaction (AKASAA), we showed that this mutant does in fact prevent protection by calcineurin.
Project description:We used HDX-MS to map novel binding sites of calcineurin on PI4KA and FAM126A. Calcineurin binds to PxIxIT and LxVP motifs on PI4KA and FAM126A indicating a novel regulatory mechanism of Pi4KA by calcineurin
Project description:Increase in the occurrence and severity of human H5N1 spillover infections resulting from dissemination of highly pathogenic avian influenza (HPAI) virus into bird and mammal populations raises concerns about HPAI adapting to become human transmissible. Studies identified hemagglutinin acid stability and receptor preference as essential molecular traits that shape host tropism. Mutations that increase hemagglutinin acid-stability and affinity for a-2,6-linked sialic acids have been shown to confer H5N1 airborne transmissibility in a ferret model. Here, we use hydrogen/deuterium-exchange mass spectrometry to dissect how airborne adaptation mutations impact dynamics during activation. Our results reveal that such mutations suppress dynamics in pH-responsive regions including the fusion peptide and receptor binding and fusion subunit interface, which would prevent premature inactivation during airborne transmission. These results for HPAI hemagglutinin identify regions that are hotspots for adaptive changes and reveal general influenza subtype-specific patterns.
Project description:Inositol pyrophosphates are highly phosphorylated nutrient messengers. The final step of their biosynthesis is catalyzed by diphosphoinositol pentakisphosphate kinase (PPIP5K) enzymes, which are conserved among fungi, plants, and animals. PPIP5Ks contain an N-terminal kinase domain that generates the active messenger 1,5-InsP8 and a C-terminal phosphatase domain that participates in PP-InsP catabolism. The balance between kinase and phosphatase activities controls the cellular levels and signaling capacity of 1,5- InsP8. Here, we present crystal structures of the apo and substrate-bound Vip1 phosphatase domain from S. cerevisiae (ScVip1PD). ScVip1PD is a phytase-like inositol 1-pyrophosphate phosphatase with two conserved histidine phosphatase catalytic motifs. The enzyme has a strong preference for 1,5-InsP8 and is inhibited by inorganic phosphate. ScVip1PD has an α-helical insertion domain stabilized by a structural Zn2+ binding site, and a unique GAF domain that exists in an open and closed state, allowing channeling of the 1,5-InsP 8 substrate to the active site. Mutations that alter the active site, that restrict the movement of the GAF domain or that modify the charge of the substrate channel significantly inhibit the activity of the yeast enzyme in vitro, and the function of the Arabidopsis PPIP5K VIH2 in planta. Structural analyses of full-length PPIP5Ks suggest that the kinase and phosphatase are independent enzymatic modules. Taken together, our work reveals the structure, enzymatic mechanism and regulation of eukaryotic PPIP5K phosphatases
Project description:Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperones promote de novo folding, suggesting that their activity is coordinated at the ribosome. Here we use biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We find that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to solvent-accessible sites in a sequence non-specific manner. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.
Project description:Hydroxyl radical protein footprinting (HRPF) is a mass spectrometry (MS)-based method for studying protein structures, interactions, conformations, and folding. We evaluated dimethylthiourea (DMTU) as a replacement for catalase in quenching HRPF reactions. We observed that the DMTU is highly effective at quenching HRPF oxidation. DMTU does not cause background protein issues like catalase, resulting in an increased number of protein identifications from complex mixtures.
Project description:LC-MS/MS analysis of a gel slice containing a disulfide-cross-linked peptide chains between thioredoxin-1 and the NACHT-LRR domains of NLRP1