Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. In order to investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation, we generated a model dataset by cross-linking human serum albumin (HSA) with the readily available cross-linker BS3-d0/d4 in different heavy/light ratios.
Project description:We have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.
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:As a part of a study on how kinetochors are assembled at the centromeres of the chromosomes, cross-linking/mass spectrometry has been applied to investigate interactions between Okp1/ Ame1 heterodimer, which is part of the COMA complex, and CENP-A from Saccharomyces cerevisiae.
Project description:Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue- resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. Wefound two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue- resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so.We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Ourwork lays the foundations of studying themolecular details of biological processes at greater ease than this could be done so far.
Project description:Budding yeast Tsr1 is an essential ribosome biogenesis factor that is required for cytoplasmic steps in 40S subunit maturation. S. cerevisiae Tsr1 was expressed as an N-terminal GST fusion protein in E. coli. A mutant Tsr1ΔNΔloop was generated in which residues 410 to 476 of Tsr1 were replaced with a short glycine and serine rich sequence; in addition, the N-ternimal 45 amino acid residues were replaced by five amino acid residues “GPDSD”. Tsr1ΔNΔloop allowed for generating native crystals that diffracted to 3.6 Å. Here we characterize both wild type Tsr1 and Tsr1ΔNΔloop using cross-linking/mass spectrometry.
Project description:The mouse PIWI-interacting RNA (piRNA) pathway provides anti-transposon immunity to the developing male germline by directing transposon DNA methylation. The first step in this process is the recruitment of SPOCD1 to young LINE1 loci 1 followed in the second step by piRNA-mediated tethering of the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript. To protect the germline, the piRNA pathway needs to methylate all active transposon copies but how this is achieved remains unknown. Here, we show that nuclear piRNA and de novo methylation factors are all euchromatic. We find that SPOCD1 directly interacts with the nuclear pore component TPR, which forms heterochromatin exclusion zones adjacent to nuclear pores. In foetal gonocytes undergoing piRNA-directed DNA methylation, TPR is found both at the nuclear periphery but also abundantly throughout the nucleoplasm. We found that the SPOCD1-TPR interaction is required for complete non-stochastic piRNA-directed LINE1 methylation. The loss of the SPOCD1-TPR interaction results in a fraction of SPOCD1 and other chromatin-bound piRNA factors to relocalise to constitutive heterochromatin where they are no longer accessible to MIWI2 and the de novo methylation machinery. We propose that TPR-mediated heterochromatin exclusion provides a nowhere-to-hide mechanism for SPOCD1-bound LINE1 loci throughout the nucleoplasm. In summary, the piRNA pathway has co-opted TPR to guarantee LINE1s are euchromatic and accessible to the piRNA and de novo methylation machineries.
Project description:We reveal that MCAK has a compact conformation in solution using cross-linking and electron microscopy. When MCAK is bound to the microtubule ends, it adopts an extended conformation with the N terminus and neck region of MCAK interacting with the microtubule. Also Aurora Bphosphorylation does not alter MCAK conformation in solution. Aurora B interferes with the extended conformation of MCAK on microtubules to decrease the affinity of MCAK for microtubules and reduces its depolymerase activity in a graded fashion.
Project description:The Fanconi Anemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks. Genetic inactivation of this pathway impairs development, prevents blood production and promotes cancer. The key molecular step in the FA pathway is the monoubiquitination of a heterodimer of FANCI-FANCD2 by the FA core complex - a megadalton multiprotein E3 ubiquitin ligase. Monoubiquitinated FANCI-FANCD2 then activates a pathway to remove the DNA crosslink. Lack of molecular insight into the FA core complex limits a detailed explanation of how this vital DNA repair pathway functions. Here we reconstituted an active, recombinant FA core complex, and used electron cryo-microscopy (cryo-EM) and mass spectrometry to determine its overall structure. The FA core complex is comprised of a central symmetric dimer of the FANCB and FAAP100 subunits, flanked by two copies of the RING finger protein, FANCL. This acts as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. The two FANCL subunits are positioned at opposite ends of the complex in an unusual asymmetric arrangement, distinct from other E3 ligases. We propose that each of the two FANCL subunits play unique roles within the complex – one is a structural component while the other monoubiquitinates FANCD2. The cryo-EM structure of the FA core complex, supported by crosslinking mass spectrometry and native mass spectrometry, therefore provides a foundation for a detailed understanding of this fundamental DNA repair pathway.