ABSTRACT: We use HDX-MS to characterise the binding interface between the tick evasin P672 and the chemokine CCL8, in order to assist in the development of peptides capable of mimicking P672 anti-inflammatory activity.
Project description:Serum amyloid A (SAA) is a plasma protein that transports lipids during inflammation. To explore SAA solution conformations and lipid binding mechanism, we used hydrogen-deuterium exchange mass spectrometry, lipoprotein reconstitution, sequence analysis and molecular dynamics simulations. Solution conformations of lipid-bound and lipid-free mSAA1 at pH~7 agreed in details with the crystal structures but also showed important differences. The results revealed that amphipathic α-helices h1 and h3 comprise a lipid-binding site that is partially pre-formed in solution, is stabilized on lipoproteins, and shows lipid-induced folding of h3. This site sequesters apolar ligands via a concave hydrophobic surface in SAA oligomers. The largely disordered C-terminal region is conjectured to mediate promiscuous binding of other ligands. The h1-h2 linker region forms an unexpected β-hairpin that may represent an early amyloidogenic intermediate. The results establish structural underpinnings for understanding SAA interactions with lipids and other ligands, its evolutional conservation, and its transition to amyloid.
Project description:General control nonderepressible 2 (GCN2) phosphorylates eIF2α, regulating translation in response to nutritional stress. Here, we show that mammalian ribosomes are potent GCN2 activators. Hydrogen/deuterium exchange–mass spectrometry (HDX-MS) showed GCN2 interacting with domain II of the uL10 P-stalk protein. The P-stalk is a uL10/P12/P22 pentameric complex that is part of the ribosomal GTPase-associated center. Recombinant human P-stalk greatly stimulates GCN2. Both domain II of uL10 and the C-terminal tails of P1 and P2 are necessary for maximal GCN2 activation. On actively translating ribosomes, the C-terminal tails of P1 and P2 are sequestered by elongation factors, suggesting P-stalk availability could link translational stress to GCN2 activation.
Project description:We present HDX-MS data of apo-SurA, SurA in complex with OmpX, OmpF and a peptide with sequence WEYIPNV to define (1) interdomain dynamics in the periplasmic chaperone SurA, and (2) the binding site of OmpX, OmpF and WEYIPNV on SurA.
Project description:Heme regulatory motifs (HRMs) are found in a variety of proteins that are involved in diverse biological functions. In the C-terminal tail region of human heme oxygenase-2 (HO2), there are two HRMs whose cysteines form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs is independent of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the core of HO2. Using HDX-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle – when Fe3+-heme is bound to the HRMs and the core is in the apo state. The conformational changes detected are consistent with transfer of heme between binding sites. Indeed, Fe3+-heme bound to the HRMs is transferred to the apo-core upon either independently expressing the core and a construct spanning the HRM-containing tail or after single turnover of heme at the core. In addition, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We thus propose a Fe3+-heme transfer model in which heme bound to the HRMs is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.
Project description:Integrins αVβ8 and αVβ6 are master activators of proTGF-βs. Whereas most integrins including αVβ6 are activated by tensile force applied by the cytoskeleton, αVβ8 links differently to the cytoskeleton and lacks the open headpiece conformation that represents the high affinity state of other integrins. Here, we shed light on the atypical activation mechanism of integrin αVβ8 using crystal structures in the absence and presence of proTGF-β1 peptide, comparisons of the hydrogen deuterium exchange dynamics of αVβ8 and αVβ6, and affinity measurements on mutants in which structurally atypical residues of αVβ8 and typical residues of αVβ6 were reciprocally exchanged.
Project description:In eukaryotes, E1 initiates the ubiquitin cascade by adenylation and thioesterification of the ubiquitin C-terminus and subsequent transfer of ubiquitin to E2 enzymes. A clinical-grade small molecule that binds to the E1 ATP binding site and covalently derivatizes the ubiquitin C-terminus effectively shuts down E1 enzymatic activity. However, mutation at or near the ATP binding site of E1 causes resistance, mandating alternative approaches to blocking what is otherwise a promising cancer target. Here, we identified a helix-in-groove interaction between the N-terminal alpha-1 helix of E2 and a pocket within the ubiquitin fold domain of E1 as a druggable site of protein interaction. By generating and optimizing stapled alpha-helical peptides (SAHs) modeled after the E2 alpha-1 helix, we achieve site-specific engagement of E1, induce a consequential conformational change, and effectively block E1 enzymatic activity, resulting in a generalized disruption of E2 ubiquitin-charging that suppresses ubiquitination of cellular proteins. Thus, we provide a blueprint for an alternative E1-targeting strategy for the treatment of cancer. Hydrogen exchange mass spectrometry was used to characterize the predominant E1 enzyme in mammals (UBE1, a 118 kDa multi-domain enzyme that catalyzes both ubiquitin adenylation and thioesterification) in the unbound state. We then interrogated the structural impact of UBE1 interaction with the stapled peptide SAH-UBE2A and several mutants. The observed peptide-induced exposure of the ubiquitin-fold domain (UFD) linker hinge in UBE1 was consistent with an inhibitory mechanism whereby SAH-UBE2A locks UBE1 into its proximal UFD conformation.
Project description:Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 (N8) from activated Cullins. The structure of stable CSN-CRL can be used to understand this mechanism of regulation. Here we present the first structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (MS). These structures reveal a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen deuterium exchange-MS we show that CRL2 binding and conformational activation of CSN5/CSN6 occur in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identified novel sensory regions of the CSN that mediate its stepwise activation mechanism and provide a framework for better understanding the regulatory mechanism of other Cullin family members.
Project description:The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.
Project description:Collagen deposition is a key process during idiopathic pulmonary fibrosis (IPF); however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analysis does not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labelled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation from gene expression analysis. Lung fibrosis was induced in female C57BL/6 mice by bleomycin instillation and sacrificed. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labelled with deuterium by providing mice with deuterated drinking water. After sacrifice, lung tissue was collected for microarray analysis, determination of new collagen formation, and histology. Deuterated water labelling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data revealed fibrosis specific processes, of which proliferation was an unexpected one. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, tenascin C, MMP-14, lysyl oxidase, and type V collagen. These data demonstrate, using a novel combination of technologies, that proliferation and extracellular matrix production are correlated to the core process of fibrosis, i.e. the formation of new collagen. In addition, it identified genes directly correlated to fibrosis, thus providing more insight into the aetiology of IPF. Total RNA was obtained from mouse lungs at timepoint 0 as a control (n = 7) or timepoints 1 (n = 7), 2 (n = 6), 3 (n = 6), 4 (n = 6) or 5 (n = 6) weeks after bleomycin-instillation to induce lung fibrosis.