Project description:Cysteine residues ubiquitously stabilize tertiary and quaternary protein structure by formation of disulfide bridges. Here we investigate another linking interaction that involves sulfhydryl groups of cysteines, namely intra- and intermolecular methylene-bridges between cysteine and lysine residues. A number of crystal structures possessing such a linkage were identified in the Protein Data Bank. Inspection of the electron density maps and re-refinement of the nominated structures unequivocally confirmed the presence of Lys-CH2 -Cys bonds in several cases.
Project description:We recently reported the discovery of a lysine-cysteine redox switch in proteins with a covalent nitrogen-oxygen-sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur-oxygen-nitrogen-oxygen-sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.
Project description:The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.
Project description:Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetyl-Lys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific antiacetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.
Project description:The modification of lysine residues with acylating agents has represented a ubiquitous approach to the construction of antibody conjugates, with the resulting amide bonds being robustly stable and clinically validated. However, the conjugates are highly heterogeneous, due to the presence of numerous lysines on the surface of the protein, and greater control of the sites of conjugation are keenly sought. Here we present a novel approach to achieve the targeted modification of lysines distal to an antibody fragment's binding site, using a disulfide bond as a temporary 'hook' to deliver the acylating agent. This cysteine-to-lysine transfer (CLT) methodology offers greatly improved homogeneity of lysine conjugates, whilst retaining the advantages offered by the formation of amide linkages.
Project description:Zero-length isopeptide crosslinks between the side chains of glutamine and lysine are the product of transglutaminase activity. It is generally accepted that transglutaminase activity is dormant under physiological conditions because the calcium concentration inside cells is too low to activate transglutaminase to an open conformation with access to the catalytic triad. Traditional assays for transglutaminase activity measure incorporation of biotin pentylamine or of radiolabeled putrescine in the presence of added calcium. In this report, we identified naturally occurring isopeptide crosslinked proteins using the following steps: immunopurification of tryptic peptides by binding to anti-isopeptide antibody 81D1C2, separation of immunopurified peptides by liquid chromatography-tandem mass spectrometry, Protein Prospector database searches of mass spectrometry data for isopeptide crosslinked peptides, and manual evaluation of candidate crosslinked peptide pairs. The most labor intense step was manual evaluation. We developed criteria for accepting and rejecting candidate crosslinked peptides and showed examples of MS/MS spectra that confirm or invalidate a possible crosslink. The SH-SY5Y cells that we examined for crosslinked proteins had not been exposed to calcium and had been lysed in the presence of ethylenediaminetetraacetic acid. This precaution allows us to claim that the crosslinks we found inside the cells occurred naturally under physiological conditions. The quantity of crosslinks was very low, and the crosslinked proteins were mostly low abundance proteins. In conclusion, intracellular transglutaminase crosslinking/transamidase activity is very low but detectable. The low level of intracellular crosslinked proteins is consistent with tight regulation of transglutaminase activity.
Project description:Post-translational modification (PTM) occurs after a protein is translated from ribonucleic acid. It is an important living creature life phenomenon because it is implicated in almost all cellular processes. Identification of PTM sites from a given protein sequence is a hot topic in bioinformatics. Lots of computational methods have been proposed, and they provide good performance. However, most previous methods can only tackle one PTM type. Few methods consider multiple PTM types. In this study, a multi-label classification model, named RMTLysPTM, was developed to recognize four types of lysine (K) PTM sites, including acetylation, crotonylation, methylation and succinylation. The surrounding sites of a lysine site were selected to constitute a peptide segment, representing the lysine at the center. Deep analysis was conducted to count the distribution of 2-residues with fixed location across the four types of lysine PTM sites. By aggregating the distribution information of 2-residues in one peptide segment, the peptide segment was encoded by informative features. Furthermore, a prediction engine that can precisely capture the traits of the above representations was designed to recognize the types of lysine PTM sites. The cross-validation results on two datasets (Qiu and CPLM training datasets) suggested that the model had extremely high performance and RMTLysPTM had strong generalization ability by testing it on protein Q16778 and CPLM testing datasets. The model was found to be generally superior to all previous models and those using popular methods and features. A web server was set up for RMTLysPTM, and it can be accessed at http://119.3.127.138/.
Project description:The S-alkylation of Cys residues with a maleimide and the Nϵ -acylation of Lys residues with an N-hydroxysuccinimide (NHS) ester are common methods for bioconjugation. Using Cys and Lys derivatives as proxies, we assessed differences in reactivity depending on the position of Cys or Lys in a protein sequence. We find that Cys position is exploitable to improve site-selectivity in maleimide-based modifications. Reactivity decreases substantially in the order N-terminal>in-chain>C-terminal Cys due to modulation of sulfhydryl pKa by the α-ammonium and carboxylate groups at the termini. A lower pKa value yields a larger fraction thiolate, which promotes selectivity while somewhat decreasing thiolate nucleophilicity in accord with β n u c =0.41. Lowering pH and salt concentration enhances selectivity still further. In contrast, differences in the reactivity of Lys towards an NHS ester were modest due to an appreciable decrease in amino group nucleophilicity with a lower pKa of its conjugate acid. Hence, site-selective Lys modification protocols will require electrophiles other than NHS esters.
Project description:Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examining crosslinks in a complex protein mixture consisting of human SH-SY5Y cells exposed to 10 μM dichlorvos. The steps in our protocol included immunopurification of crosslinked peptides by binding to anti-isopeptide antibody 81D1C2, stringent washing of the immobilized complex, release of bound peptides from Protein G agarose with 50% acetonitrile 1% formic acid, liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer, Protein Prospector searches of mass spectrometry data, and manual evaluation of candidate crosslinked dipeptides. We report a low quantity of dichlorvos-induced KD and KE crosslinked proteins in human SH-SY5Y cells exposed to dichlorvos. Cells not treated with dichlorvos had no detectable KD and KE crosslinked proteins. Proteins in the crosslink were low abundance proteins. In conclusion, we provide a protocol for testing complex protein mixtures for the presence of crosslinked proteins. Our protocol could be useful for testing the association between neurodegenerative disease and exposure to organophosphorus pesticides.
Project description:Targeted covalent inhibitor design is gaining increasing interest and acceptance. A typical covalent kinase inhibitor design targets a reactive cysteine; however, this strategy is limited by the low abundance of cysteine and acquired drug resistance from point mutations. Inspired by the recent development of lysine-targeted chemical probes, we asked if nucleophilic (reactive) catalytic lysines are common on the basis of the published crystal structures of the human kinome. Using a newly developed p Ka prediction tool based on continuous constant pH molecular dynamics, the catalytic lysines of eight unique kinases from various human kinase groups were retrospectively and prospectively predicted to be nucleophilic, when kinase is in the rare DFG-out/αC-out type of conformation. Importantly, other reactive lysines as well as cysteines at various locations were also identified. On the basis of the findings, we proposed a new strategy in which selective type II reversible kinase inhibitors are modified to design highly selective, lysine-targeted covalent inhibitors. Traditional covalent drugs were discovered serendipitously; the presented tool, which can assess the reactivities of any potentially targetable residues, may accelerate the rational discovery of new covalent inhibitors. Another significant finding of the work is that lysines and cysteines in kinases may adopt neutral and charged states at physiological pH, respectively. This finding may shift the current paradigm of computational studies of kinases, which assume fixed solution protonation states.