Cross-linked ammonia monooxygenase complex in BN-PAGE gel from Nitrososphaera viennensis
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ABSTRACT: A band containing the ammonia monooxygenase complex from Nitrosospheara viennensis was cut from a Blue Native PAGE gel and cross linked using DSSO.
Project description:human OGDH (E1o) and DHTKD1 (E1a) were cross-linked with DLST (E2o). The samples were analyzed by LC-MS/MS in order to elucidate the interaction sites between these two E1-E2 subcomplexes.
Project description:CL-MS OF hE1a (DHTKD1) or G729R hE1a with hE2o to identify the possible difference in the interactions between hE1a-hE2o and G729R hE1a-hE2o
Project description:Disuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has been applied to multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas-phase. In this study, we further advance the structural capabilities of DSBU by twisting the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS-cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl dibutyric imide (DSBI) as prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography (IMAC) enrichment. We detail DSBI synthesis and describe its behavior in solution and in the gas-phase while cross-linking isolated proteins and human cell lysates. DSBI and DSBU cross-links are compared at the same enrichment depths to bridge these two cross-linker classes. We validate DSBI cross-links by mapping them in high-resolution structures of large protein assemblies . The cross-links observed yield insights into the morphology of intrinsically disordered proteins (IDPs) and their complexes. The DSBI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers
Project description:The endogenous cellular prion protein (PrPC) can misfold into the scrapie isoform (PrPSc) and cause fatal infectious diseases. Despite significant research on the prion protein, both its normal function and whether alterations to that function play a critical role in prion diseases remain unknown. The protein consists of a predominantly alpha-helical C-terminal domain and an unstructured N-terminal domain that can coordinate Cu2+. Previous studies using NMR and EPR have revealed a tertiary association between the N-terminal domain and the C-terminal domain that we have hypothesized to be critical to the protein’s normal function. Here we investigated and quantified the inter-domain interactions within three different prion variants (wild type recombinant mouse PrPC, mutant delta central region (ΔCR), and disease mutant (E199K) after chemical cross-linking with a newly designed MS-cleavable reagent 1-(4-((2,5-Dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-4-(2-(3-methyl-3H-diazirin-3-yl)ethyl)-1,4-diazabicyclo[2.2.2] octane-1,4-diium (APDC4), followed by nHPLC(RP) and tandem MS analysis.
Project description:Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel MS-cleavable reagents. These cross-linkers enhance the reliability of cross-link identification. Presently, only water-insoluble MS-cleavable cross-linkers are commercially available. However, the comprehensive analysis of the various chemical and structural motifs inherent in proteins depends on the targeting of different protein sites with varying degrees of hydrophilicity. We introduce a new MS-cleavable cross-linker called disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group. It therefore serves as a water-soluble counterpart to the commercially available and widely used disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers—bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU on bovine serum albumin. We also compared efficacy of DSBU and DSSBU on human hemoglobin. Our results demonstrate that the superior water solubility of DSSBU enables to avoid the usage of polar solvents such as DMSO but still maintaining the effectivity of MS-cleavable crosslinker such as DSBU.
Project description:DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigations involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA+ family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries. In the present study, we identified DPCD as a new R1R2 partner in vivo. We show that DPCD interacts directly with R1 and R2 in vitro and in cells. We characterized the physico-chemical properties of DPCD in solution and built 3D structural models of DPCD alone and in complex with R1R2 validated by experimental data combining Small-Angle X-ray Scattering and Cross-linking Mass Spectrometry, among others. Interestingly, DPCD disrupts the dodecameric state of R1R2 complex upon binding and this interaction occurs mainly via the DII domains of R1R2.
Project description:LRRK2 serine/threonine kinase is associated with inherited Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective and closely related PPM1J/M exhibit virtually no activity toward substrates such as Rab8a phosphorylated at T72 (pT72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a conserved catalytic domain that adopts a β-sandwich fold. The striking difference is that PPM1H has evolved a 110-residue flap domain that punctuates the catalytic domain. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimera with the PPM1H flap domain dephosphorylates pT72 of Rab8a with a higher specific activity than PPM1H. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold.
Project description:Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.
Project description:We applied quantitative cross-linking/mass spectrometry (QCLMS) to interrogate the structure of iC3 (or C3(H2O)), the activated hydrolytic product of the abundant human complement protein C3. The slow but spontaneous and ubiquitous formation of iC3 from C3 initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. QCLMS revealed structural differences and similarities between iC3 and C3, as well as between iC3 and C3b that is a pivotal proteolytic cleavage product of C3 and is functionally similar to iC3. Considered in combination with the crystal structures of C3 and C3b, our data support a model wherein the thioester-containing domain of C3 swings to the other end of the molecule creating, in iC3, a stable C3b-like platform for binding the zymogen, factor B, or the regulator, factor H. The integration of available crystallographic and QCLMS data allowed the determination of a 3D model for iC3. The unique arrangement of domains in iC3, which retains the anaphylatoxin (ANA) domain (while ANA is excised when C3 is enzymatically activated to C3b), is consistent with observed differences in activation and regulation between iC3 and C3b.
Project description:Chemical cross-linking coupled with mass spectrometry (CXMS) offers the distance constraints critical for building the structural model of protein and protein complexes and understanding dynamics of biological systems. Originally developed for protein structural models, CXMS has evolved into method for studying protein complex formation, ligand-induced conformational changes, and quantitative structural analysis using isotopically labeled cross-linkers. In this study we tested the potential of isotopically labelled MS-cleavable cross-linker to track the stability of the therapeutic monoclonal antibodies. A novel isotopically labelled MS-cleavable cross-linker was synthesized, and its reactivity was successfully tested on peptide and protein standards. Further, the novel cross-linker was utilized to test the stability of selected therapeutical monoclonal antibodies, Avastin and Herceptin, adopting the data-independent acquisition. This study reports the advantages of using combination of 13C isotopically labelled MS-cleavable cross-linkers and data-independent mass spectrometry analysis for the automated identification of cross-linked products and thus monitoring the structural rearrangement of protein structure.