Project description:we wanted to see where and how the protein p37 interacts with its partner proteins p97, PPI,Sds22 and I3. To this end p37 derivatives with genetically encoded crosslinking amino acid (p-benzoyl-L-phenylalanine) were generated. After incubation with the partner proteins, photocross-linking and tryptic digest the resulting cross-linked peptides were analysed by mass spectrometry.
Project description:We wanted to see where and how the protein p97 interacts with its partner proteins p37, PPI,Sds22 and I3. To this end a p97 derivative with genetically encoded crosslinking amino acid (p-benzoyl-L-phenylalanine) was generated. After incubation with the partner proteins, photocross-linking and tryptic digest the resulting cross-linked peptides were analysed by mass spectrometry.
Project description:ATP-sensitive potassium (K-ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic beta-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse K-ATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of K-ATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. CyanurBiotinDimercaptoPropionylSuccinimide (CBDPS) cross-linking mass spectrometry was used to partially confirm cryoEM structures.
Project description:Histone modifications perform a vast array of functions in regulating gene expression, DNA replication, and repair. Monoubiquitination of histone H2B at K123 in yeast (K120 in humans) is an intriguing modification because it is deposited cotranscriptionally, mediates the installation of several other epigenetic marks, and then disappears; hence, it is associated transiently with actively transcribed chromatin. In yeast, the H2B ubiquitin writer is the E2/E3 pair Rad6/Bre1, and there are two deubiquitinases that can erase it, Ubp8 and Ubp10. Whilst Ubp8 resides within the larger SAGA complex, Ubp10 (USP36 in humans) is a monomeric and constitutively active deubiquitinase, raising questions as to what processes regulate it, given it would be undesirable for H2B to be deubiquitinated prematurely before downstream processes connected to this epigenetic mark occur. Here we show that Ubp10’s activity is regulated by acidic regions within its long N-terminal intrinsically disordered region (IDR), which extensively interact with H2A/H2B dimers, as shown by crosslinking mass spectrometry. These interactions vanish when H2A/H2B is present in nucleosomes. These observations explain why Ubp10 has low baseline activity on nucleosomes, but is activated by FACT, a histone chaperone which evicts H2A/H2B dimers from nucleosomes, thereby generating Ubp10’s preferred substrate, which we demonstrate with single molecule fluorescence experiments. Hence, this work provides a biophysical mechanism for how Ubp10 can provide a housekeeping function to deubiquitinate actively-transcribed DNA, wherein FACT produces a temporary pool of H2A/H2B dimers.
Project description:Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. The Chd1 remodeler specializes in shifting nucleosomes into evenly spaced arrays, a defining characteristic of chromatin in gene bodies that blocks spurious transcription initiation. Linked to some forms of autism and commonly mutated in prostate cancer, Chd1 is essential for maintaining pluripotency in stem cells. Here we report a complex of yeast Chd1 bound to a nucleosome in a nucleotide-free state, determined by cryo-electron microscopy (cryo-EM) to 2.6 Å resolution. The structure shows a bulge of the DNA tracking strand where the ATPase motor engages the nucleosome, consistent with an initial stage in DNA translocation. Unlike other remodeler-nucleosome complexes, nucleosomal DNA compensates for the remodeler-induced bulge with a bulge of the complementary DNA strand one helical turn downstream from the ATPase motor. Unexpectedly, the structure also reveals an N-terminal binding motif, called ChEx, which binds on the exit-side acidic patch of the nucleosome. The ChEx motif can displace a LANA-based peptide from the acidic patch, which suggests a means by which Chd1 remodelers may block competing chromatin remodelers from acting on the opposite side of the nucleosome.
Project description:The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a novel p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved novel -helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.
Project description:Here we present data using chemical crosslinking to inform on the architecture of the SPATA5-SPATA5L1-C1orf109-CINP complex (or the 55LCC).
Project description:To investigate the structural basis of SurA’s chaperone function, we characterized crosslinks between it and two of its clients, OmpA and OmpX, and used those as distance restraints to build structural models.
Project description:Cross-linking mass spectrometry has evolved as a powerful technique to study protein-protein interactions and to provide structural information over the past decades. Low reaction efficiencies, and complex matrices lead to challenging system wide crosslink analysis. In this study, we improved and streamlined an Azide-A-DSBSO based in vivo crosslinking workflow employing two orthogonal effective enrichment steps: Affinity enrichment and size exclusion chromatography (SEC). Combined, they allow an effective pulling of DSBSO containing peptides and remove the background of linear as well as mono-linked peptides. We found that the analysis of a single SEC fraction is effective to yield ~90% of all crosslinks, which is important whenever measurement time is limited, and sample throughput is crucial. Our workflow resulted in more than 5000 crosslinks from K562 cells and generated a comprehensive PPI network from whole cells as well as nuclear extracts. From 393 PPI found within the nucleus, 56 have not yet been reported in the STING database and are representing a valuable resource for investigating new molecular mechanisms and provide complementary data for future studies. We further show, that by applying DSBSO to nuclear extracts we yield more crosslinks on lower abundant proteins and showcase this on the DEAD-box RNA helicase DDX39B which is predominantly expressed in the nucleus. Our data indicates that DDX39B is present in monomeric and dimeric form together with DDX39A within the nuclear extracts analyzed.