Project description:In this study, we employed the photoactivatable crosslinker (sulfo-SDA) to investigate protein-protein interaction between human Separase-Scc1 fusion protein and SMC1/SMC3 cohesin subunits complex in the presence of DNA.
Project description:The influenza nuclear export protein (NEP)/non-structural protein 2 (NS2) is a multifunctional protein, involved in viral ribonucleoprotein (vRNP) export from the nucleus, genome replication enhancement, and the adaption of avian influenza to mammals. Despite the growing attention on the importance of NEP in the influenza lifecycle and in interspecies transmission from avian hosts, the molecular details of how it is able to perform its various roles is still yet to be fully understood. In the interest of generating antiviral proteins and to aid in structural characterization, a panel of Rep proteins were raised against influenza virus A NEPH1N1. When complexed with RepE4 we were able to crystallize full-length NEPH1N1, revealing a folded and monomeric conformation. The N- and C-termini of NEPH1N1 pack together, with the middle linker region resembles a hinge, con-trasting a previous structure where NEP is dimeric and elongated. Together these structures demonstrate the plasticity of NEP, a trait which may potentially aid NEP in binding a diversity of cellular and viral partners. Using isothermal titration calorimetry (ITC) we measured a nanomolar interaction between RepE4 and NEP. Similarly, we found that RepE4 can also bind NEP from hu-man infecting avian H7N9 and bovine originating avian H5N1 influenza viruses. Owing to their high degree of conservation, RepE4 likely has the capacity to interact with NEP from numerous influenza A virus strains. Indeed, this, combined with the nanomolar affinity measured between NEP/RepE4 could be explored further as a broad-range therapeutic strategy and/or a tool in cellulo to understand NEP function at the molecular level.
Project description:mRNA decay is a key determinant of gene regulation, initiated by the shortening of mRNA poly(A) tails by the CCR4-NOT deadenylation complex. Specificity is achieved through RNA adaptors—RNA-binding proteins that recruit CCR4-NOT to specific substrate mRNAs in a regulated manner. However, the molecular mechanisms underlying this specificity remain unclear in many cases. In this study, we applied crosslinking MS to investigate the interaction between the fission yeast RNA-binding protein Puf3 and the Ccr4-Not complex.
Project description:We introduce a complimentary, heterobifunctional, photoactivatable, benzophenone containing cross-linker and show its successful application to cross-linking/mass spectrometry, by increasing data density, when used alongside a previously developed diazirine-based heterobifunctional cross-linker.
Project description:The low density lipoprotein receptor-related protein 2 (LRP2 or megalin) is a multiligand endocytic receptor implicated in the homeostasis of several organs. Mutations in the LRP2 gene are associated with severe systemic disease. It is well-know that the low density lipoprotein receptor-related protein-associated protein 1 (LRPAP1 or RAP) interacts with LRP2 modulating its function. A single copy of LRPAP1 was shown to interact with complement-type repeats in LRP2, however this domain is highly redundant in LRP2, hinting at a different stoichiometry of the complex. Here, using cryogenic-electron microscopy, AlphaFold and cross-linking mass spectrometry, we provide structural insights into human recombinant LRP2 in the presence and absence of LRPAP1. Our integrative approach reveals three additional LRPAP1 sites in LRP2. Some of these LRPAP1 binding regions overlap with ligand binding sites. This finding, supported by competitive in vitro binding assays supports the hypothesis that LRPAP1 acts as a direct modulator of LRP2's ligand-binding activity. In addition, we identify several pathogenic LRP2 mutations located at the LRP2-LRPAP1 interface and LRPAP1 binding regions unique to LRP2 within the LRP family. Taken together, our study provides a broader molecular landscape of LRP2-LRPAP1 interactions, offering insights into its clinical and functional role.
Project description:The low density lipoprotein receptor-related protein 2 (LRP2 or megalin) is a multiligand endocytic receptor implicated in the homeostasis of several organs. Mutations in the LRP2 gene are associated with severe systemic disease. It is well-know that the low density lipoprotein receptor-related protein-associated protein 1 (LRPAP1 or RAP) interacts with LRP2 modulating its function. A single copy of LRPAP1 was shown to interact with complement-type repeats in LRP2, however this domain is highly redundant in LRP2, hinting at a different stoichiometry of the complex. Here, using cryogenic-electron microscopy, AlphaFold and cross-linking mass spectrometry, we provide structural insights into human recombinant LRP2 in the presence and absence of LRPAP1. Our integrative approach reveals three additional LRPAP1 sites in LRP2. Some of these LRPAP1 binding regions overlap with ligand binding sites. This finding, supported by competitive in vitro binding assays supports the hypothesis that LRPAP1 acts as a direct modulator of LRP2's ligand-binding activity. In addition, we identify several pathogenic LRP2 mutations located at the LRP2-LRPAP1 interface and LRPAP1 binding regions unique to LRP2 within the LRP family. Taken together, our study provides a broader molecular landscape of LRP2-LRPAP1 interactions, offering insights into its clinical and functional role.
Project description:The low density lipoprotein receptor-related protein 2 (LRP2 or megalin) is a multiligand endocytic receptor implicated in the homeostasis of several organs. Mutations in the LRP2 gene are associated with severe systemic disease. It is well-know that the low density lipoprotein receptor-related protein-associated protein 1 (LRPAP1 or RAP) interacts with LRP2 modulating its function. A single copy of LRPAP1 was shown to interact with complement-type repeats in LRP2, however this domain is highly redundant in LRP2, hinting at a different stoichiometry of the complex. Here, using cryogenic-electron microscopy, AlphaFold and cross-linking mass spectrometry, we provide structural insights into human recombinant LRP2 in the presence and absence of LRPAP1. Our integrative approach reveals three additional LRPAP1 sites in LRP2. Some of these LRPAP1 binding regions overlap with ligand binding sites. This finding, supported by competitive in vitro binding assays supports the hypothesis that LRPAP1 acts as a direct modulator of LRP2's ligand-binding activity. In addition, we identify several pathogenic LRP2 mutations located at the LRP2-LRPAP1 interface and LRPAP1 binding regions unique to LRP2 within the LRP family. Taken together, our study provides a broader molecular landscape of LRP2-LRPAP1 interactions, offering insights into its clinical and functional role.
Project description:FocA belongs to the widespread, evolutionarily ancient formate-nitrite transporter 30 (FNT) family of pentameric anion channels and translocates formic acid bidirectionally. 31 Here, we identify compartmentalized polarity distribution across the complete FocA 32 pore structure – resolved at 2.56 Å – mirrored against a two-fold axis with H209 at its 33 center. The FocA-H209N efflux-only variant reveals a density consistent with formic 34 acid located directly at N209, breaking local polarity distribution. Pyruvate formate-35 lyase, generating formate, orients at the cytoplasmic face where formate delivery is 36 regulated by conformational changes in the FocA vestibule. Comparisons with other 37 FNTs suggest a tuning mechanism of formate-specific transport via checkpoints 38 enriched in hydrophilic residues.
Project description:The ring-shaped cohesin complex is thought to fulfil its roles in sister chromatid cohesion, genome stability and gene regulation by topologically encircling DNAs. The ring is formed by two Structural Maintenance of Chromosome (SMC) subunits, whose ATPase heads are linked by a kleisin subunit. Additional components, including the Mis4Scc2/NIPL cohesin loader, engage the kleisin. We applied crosslinking mass spectrometry to characterize cohesion complex in two conditions: initial binding and gripping state.
Project description:[NiFe]-hydrogenases catalyse the reversible splitting of hydrogen. The catalytic metal centre (NiFe(CN)2CO) is unique in biology, and assembled by an intricate protein machinery, in a process that is still being explored. We hypothesised a structural, ATP-dependent, mechanistic explanation for the assembly of the Fe(CN)2CO fragment via the HypCD complex. We carried out a crosslinking mass spectrometry (crosslinking MS) analysis to study the structure of the HypCD complex, both with (holoprotein) and without (apoprotein) the metal cofactor, and both with and without the addition of ATP. We used the UV-photoactivatable crosslinking reagent sulfo-SDA, which has been shown to have excellent performance when studying dynamic and flexible protein complexes. For photoactivation we used a high-powered LED which enabled the use of exceptionally short reaction times (20 seconds) and gave strikingly clear results. From the resulting crosslinked residue pair patterns identified, we were able to unambiguously distinguish between holoprotein with and without ATP. Crosslinks found in holoprotein, in the absence of ATP, suggested a “closed” protein conformation. When the HypCD holoprotein was crosslinked in the presence of ATP, two distinct crosslink bands were almost entirely absent, indicating that the protein conformation had shifted to an “open” conformation. Interestingly, no shift in protein conformation was evident in the crosslinked apoprotein, which implied that the cofactor was central to protein conformational dynamics. Crosslinking MS data helped to explain, and was in agreement with, protein structures predicted by AlphaFold2 (which were subsequently refined by density functional theory (DFT) modeling for placing the cofactor). Considering all the experimental data from this study led to the conclusion that the binding of ATP alone, not its hydrolysis, is required for the transfer of the Fe(CN)2CO fragment to the apo-hydrogenase large subunit.