Proteome-wide identifying site non-specific cross-linking peptides with SpotLink
Ontology highlight
ABSTRACT: We developed SpotLink to identify site non-specific cross-links from the proteome scale. Here described the Abeta dataset and the HeLa dataset to evaluate SpotLink.
Project description:Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organisation of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modelling allowed us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.
Project description:T cell receptors (TCRs) recognize specific peptides presented by human leukocyte antigens (HLAs) on the surface of antigen presenting cells and are involved in fighting pathogens and cancer surveillance. Canonical docking orientation of TCRs to their target peptide-HLAs (pHLAs) is essential for T cell activation, with reverse binding TCRs lacking functionality. TCR binding geometry and molecular interaction footprint with pHLAs is typically obtained by determining the crystal structure. Here, we describe the use of a cross-linking tandem mass spectrometry (XL-MS/MS) method to decipher the binding orienta-tion of several TCRs to their target pHLAs. Cross-linking sites were localized to specific residues and their molecular interac-tions showed differentiation between TCRs binding in canonical or reverse orientations. Structural prediction and crystal structure determination of two TCR-pHLA complexes validated these findings. The XL-MS/MS method described herein offers a faster and simpler approach for elucidating TCR-pHLA binding orientation and interactions.
Project description:High-density cross-linking/mass spectrometry data was provided for four targets (T0957, T0968, T0975 and T0987) in the CASP13 experiment (https://www.predictioncenter.org/casp13/index.cgi).
Project description:Elongator is a tRNA-modifying complex regulating protein translation. Recently, a moonlighting function of Elongator has been identified in regulating the polarization of the microtubule cytoskeleton during asymmetric cell division. Elongator induces symmetry breaking of the anaphase midzone by selectively stabilizing microtubules on one side of the spindle, contributing to the downstream polarized segregation of cell-fate determinants and therefore to cell fate determination. Here, we show how Elongator controls microtubule dynamics. Elongator binds simultaneously to the tip of microtubules and also to free GTP-tubulin heterodimers using two different subcomplexes, Elp123 and Elp456, respectively. We show that these activities must be coupled for Elongator to decrease the critical concentration of tubulin for microtubule elongation. As a consequence, Elongator increases microtubule growth speed and decreases their catastrophe rate. Surprisingly, we found that Elp456 binds to tubulin tails and has strong selectivity towards polyglutamylated tubulin. Hence, microtubules assembled by Elongator become selectively enriched with polyglutamylated tubulin. Therefore, Elongator rewrites the tubulin code of growing microtubules, placing it at the core of cytoskeletal dynamics and polarization during asymmetric cell division.
Project description:Cross-Linking/mass spectrometry draws structural information out of covalently linked peptide pairs. When these links do not match to previous structural models they may indicate changes of protein conformation. Unfortunately, such links can also be the result of experimental errors or artefacts. Here we describe the observation of non-covalently associated peptides during liquid chromatography-mass spectrometry analysis which can easily be misidentified as cross-linked. Strikingly, they often mismatch to the protein structure or may falsely suggest protein homo-dimerization. Non-covalently associated peptides presumably form during ionization and can be distinguished from cross-linked peptides by observing co-elution of the corresponding linear peptides in MS1, as well as the presence of the individual peptide fragments in mixed MS2 spectra. Interestingly, non-covalently associated peptides seem sensitive to ionization source design as we see them more prevalently on a Q Exactive than on an Orbitrap Velos mass spectrometer. Finally we show how more disruptive ionization settings such as moderate in-source fragmentation suppresses their presence.
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:We report the first blind test on the readiness of combining high-density cross-linking/mass spectrometry data in conjunction with ab initio structure prediction, through the help of Critical Assessment of protein Structure Prediction (CASP). This blind test was coordinated by the CASP organizers and utilized the experimental protocol developed in our lab for providing high-density cross-linking/mass spectrometry data in a matter of days. The CASP organizing committee identified and acquired suitable targets and published resulting data on the CASP web page for the community of structure predictors. We provided high-density cross-linking/mass spectrometry data for four targets in CASP11, revealing to us some of the current limitations of cross-linking. These are areas where the method must now develop. With CASP taking place biannually into the future, blind testing low-resolution structure analysis tools is a worthwhile and feasible undertaking.
Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18a, Mis18b and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18a, 2 Mis18b and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18a can associate with centromeres and deposit CENP-A independently of Mis18b, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.
Project description:We have implemented the use of a heterobifunctional, UV photoactivatable cross-linker, which greatly increases the number of identified cross-links compared with homobifunctional, NHS-ester based cross-linkers. We have cross-linked human serum albumin in the context of human blood serum. We present a novel methodology that combines the use of this high-resolution cross-linking with conformational space search to investigate the structure of proteins in their native environment.
Project description:The tumor suppressor protein p53 is a transcription factor that is referred to as the “guardian of the genome” and plays an important role in cancer development. P53 is active as a tetramer; the S100β homodimer binds to the intrinsically disordered C-terminus of p53, affecting its transcriptional activity. The p53/S100β complex is regarded as highly promising therapeutic target in cancer. It has been suggested that S100β exerts its oncogenic effects by altering the p53 oligomeric state. Our aim was to study the structures and oligomerization behavior of different p53/S100β complexes by electrospray ionization mass spectrometry (ESI-MS), cross-linking mass spectrometry (XL-MS), and surface plasmon resonance (SPR). For this, wild-type p53 and single amino acid variants, representing different oligomeric states of p53 (tetrameric wild-type, dimeric L344A variant, and monomeric L344P variant) were individually investigated regarding their binding behavior towards S100β. The stoichiometry of the different p53/S100β complexes were determined by ESI-MS showing that tetrameric, dimeric, and monomeric p53 variants all bind to an S100β dimer. In addition, XL-MS revealed the topologies of the p53/S100β complexes to be independent of p53’s oligomeric state. With SPR, the thermodynamic parameters were determined for S100β binding to tetrameric, dimeric or monomeric p53 variants. Our data prove that the S100β homodimer binds to different oligomeric states of p53 with identical stoichiometries and similar binding affinities. This emphasizes the need for alternative explanations to describe the molecular mechanisms underlying p53/S100β interaction.