RESUMO
Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers.
Assuntos
Proteínas 14-3-3 , Proteínas Proto-Oncogênicas c-mdm2 , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismoRESUMO
Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.
Assuntos
Fator de Iniciação 4A em Eucariotos , Esteróis , Fator de Iniciação 4A em Eucariotos/metabolismo , Espectrometria de Massas , Metano/análogos & derivados , Biossíntese de ProteínasRESUMO
Cyanobacteria have evolved over billions of years to adapt and survive in diverse climates. Environmentally, this presents a huge challenge because cyanobacteria can now rapidly form algae blooms that are detrimental to aquatic life. In addition, many cyanobacteria produce toxins, making them hazardous to animals and humans that they encounter. Rapid identification of cyanobacteria is essential to monitor and prevent toxic algae blooms. Here, we show for the first time how native mass spectrometry can quickly and precisely identify cyanobacteria from diverse aquatic environments. By monitoring phycobiliproteins, abundant protein complexes within cyanobacteria, simple, easy-to-understand mass spectral "fingerprints" were created that were unique to each species. Moreover, our method is 10-fold more sensitive than the current MALDI-TOF mass spectrometric methods, meaning that cyanobacteria can be monitored using this technology prior to bloom formation. Together, the data show great promise for the simultaneous detection and identification of co-existing cyanobacteria in situ.
Assuntos
Cianobactérias , Toxinas Biológicas , Animais , Eutrofização , Humanos , Espectrometria de MassasRESUMO
Native mass spectrometry is now an important tool in structural biology. Thus, the nature of higher protein structure in the vacuum of the mass spectrometer is an area of significant interest. One of the major goals in the study of gas-phase protein structure is to elucidate the stabilising role of interactions at the level of individual amino acid residues. A strategy combining protein chemical modification together with collision induced unfolding (CIU) was developed and employed to probe the structure of compact protein ions produced by native electrospray ionisation. Tractable chemical modification was used to alter the properties of amino acid residues, and ion mobility-mass spectrometry (IM-MS) utilised to monitor the extent of unfolding as a function of modification. From these data the importance of specific intramolecular interactions for the stability of compact gas-phase protein structure can be inferred. Using this approach, and aided by molecular dynamics simulations, an important stabilising interaction between K6 and H68 in the protein ubiquitin was identified, as was a contact between the N-terminus and E22 in a ubiquitin binding protein UBA2.
Assuntos
Aminoácidos , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Simulação de Dinâmica Molecular , UbiquitinaRESUMO
Covalent footprinting of proteins using reactive intermediates such as radicals and carbenes is emerging as a valuable tool for mapping surface accessibility, and hence binding sites of proteins. The approach generates a significant amount of mass spectrometry (MS) data, which can be time-consuming to process manually. PepFoot, a software package that allows semiautomated processing of MS data from footprinting experiments, is described. By using the open source .mz5 file format, it is able to accept data from all the major instrument manufacturers. Following manual user interrogation of one data file within a user-friendly GUI, the software then automates determination of the degree of fractional modification ( fm) with the footprinting agent across a batch of experimental data. This greatly increases efficiency and throughput compared to manual analysis of each file, and provides initial scrutiny and confidence compared to fully automated analysis. Histogram plots of fm for each peptide from the footprinted protein may be displayed within PepFoot and mapped onto an imported protein structure to reveal differential labeling patterns and hence binding sites. The software has been tested on data from carbene and hydroxyl radical labeling experiments to demonstrate its broad utility. PepFoot is released under the LGPL version 3 license, and is available for Windows, MacOS, and Linux systems at github.com/jbellamycarter/pepfoot .
Assuntos
Pegadas de Proteínas/métodos , Software , Automação Laboratorial , Sítios de Ligação , Radical Hidroxila/química , Armazenamento e Recuperação da Informação , Espectrometria de Massas/métodos , Metano/análogos & derivados , Metano/químicaRESUMO
Cyanobacteria, also known as blue-green algae, are ubiquitous organisms on the planet. They contain tremendous protein machineries that are of interest to the biotechnology industry and beyond. Recently, the number of annotated cyanobacterial genomes has expanded, enabling structural studies on known gene-coded proteins to accelerate. This review focuses on the advances in mass spectrometry (MS) that have enabled structural proteomics studies to be performed on the proteins and protein complexes within cyanobacteria. The review also showcases examples whereby MS has revealed critical mechanistic information behind how these remarkable machines within cyanobacteria function.
Assuntos
Cianobactérias , Proteômica , Proteômica/métodos , Cianobactérias/metabolismo , Biotecnologia/métodos , Proteínas de Bactérias/metabolismo , Espectrometria de Massas/métodosRESUMO
Blue-green algae, also known as cyanobacteria, contain some of the most efficient light-harvesting complexes known. These large, colourful complexes consist of phycobiliproteins which are extremely valuable in the cosmetics, food, nutraceutical and pharmaceutical industries. Additionally, the colourful and fluorescent properties of phycobiliproteins can be modulated by metal ions, making them highly attractive as heavy metal sensors and heavy metal scavengers. Although the overall quenching ability metal ions have on phycobiliproteins is known, the mechanism of heavy metal binding to phycobiliproteins is not fully understood, limiting their widespread quantitative applications. Here, we show using high-resolution native mass spectrometry that phycobiliprotein complexes bind metal ions in different manners. Through monitoring the binding equilibria and metal-binding stoichiometry, we show in particular copper and silver to have drastic, yet different effects on phycobiliprotein structure, both copper and silver modulate the overall complex properties. Together, the data reveals the mechanisms by which metal ions can modulate phycobiliprotein properties which can be used as a basis for the future design of metal-related phycobiliprotein applications.
Assuntos
Cianobactérias , Ficobiliproteínas , Cobre/metabolismo , Cianobactérias/metabolismo , Ficobiliproteínas/química , Ficobiliproteínas/metabolismo , Prata/metabolismoRESUMO
Ferric chelates like ferric tyrosinate (TYPLEX) and the closely related ferric quinate (QPLEX) are structural mimics of bacterial siderophores. TYPLEX has been trialled as a feed additive in farming of commercial broilers, reducing Campylobacter loads by 2-3 log10 and leading to faster growth and better feed consumption. These ferric chelates offer a good alternative feed additive to antibiotics helping to reduce the indiscriminate use of preventative antibiotics in broiler farming to control Campylobacter infections. In this study, we show that QPLEX binds to the Major Outer Membrane Protein (MOMP) of C. jejuni NCTC11168. MOMP is an essential and abundant outer membrane porin on the surface of the bacteria, acting as an adhesin to help establish infection by mediating attachment of C. jejuni onto the gut epithelium of broilers and establish infection. Using carbene footprinting, we map the MOMP-QPLEX interaction and show by complementary in silico docking that QPLEX enters the porin channel through interactions at the extracellular face, translocates down the channel through a dipole transverse electric field towards the opposite end and is released into the periplasm at the intracellular face of MOMP. Our studies suggest a potential mechanism for the non-antibiotic anti-Campylobacter activity of these ferric chelates.
RESUMO
Liquid extraction surface analysis (LESA) coupled to native mass spectrometry (MS) presents unique analytical opportunities due to its sensitivity, speed, and automation. Here, we examine whether this tool can be used to quantitatively probe protein-ligand interactions through calculation of equilibrium dissociation constants (Kd values). We performed native LESA MS analyses for a well-characterized system comprising bovine carbonic anhydrase II and the ligands chlorothiazide, dansylamide, and sulfanilamide, and compared the results with those obtained from direct infusion mass spectrometry and surface plasmon resonance measurements. Two LESA approaches were considered: In one approach, the protein and ligand were premixed in solution before being deposited and dried onto a solid substrate for LESA sampling, and in the second, the protein alone was dried onto the substrate and the ligand was included in the LESA sampling solvent. Good agreement was found between the Kd values derived from direct infusion MS and LESA MS when the protein and ligand were premixed; however, Kd values determined from LESA MS measurements where the ligand was in the sampling solvent were inconsistent. Our results suggest that LESA MS is a suitable tool for quantitative analysis of protein-ligand interactions when the dried sample comprises both protein and ligand.
Assuntos
Inibidores da Anidrase Carbônica , Extração Líquido-Líquido , Animais , Inibidores da Anidrase Carbônica/análise , Bovinos , Ligantes , Extração Líquido-Líquido/métodos , Espectrometria de Massas/métodos , Proteínas/química , SolventesRESUMO
Protein-protein interactions (PPIs) are key therapeutic targets. Most PPI-targeting drugs in the clinic inhibit these important interactions; however, stabilising PPIs is an attractive alternative in cases where a PPI is disrupted in a disease state. The discovery of novel PPI stabilisers has been hindered due to the lack of tools available to monitor PPI stabilisation. Moreover, for PPI stabilisation to be detected, both the stoichiometry of binding and the shift this has on the binding equilibria need to be monitored simultaneously. Here, we show the power of native mass spectrometry (MS) in the rapid search for PPI stabilisers. To demonstrate its capability, we focussed on three PPIs between the eukaryotic regulatory protein 14-3-3σ and its binding partners estrogen receptor ERα, the tumour suppressor p53, and the kinase LRRK2, whose interactions upon the addition of a small molecule, fusicoccin A, are differentially stabilised. Within a single measurement the stoichiometry and binding equilibria between 14-3-3 and each of its binding partners was evident. Upon addition of the fusicoccin A stabiliser, a dramatic shift in binding equilibria was observed with the 14-3-3:ERα complex compared with the 14-3-3:p53 and 14-3-3:LRRK2 complexes. Our results highlight how native MS can not only distinguish the ability of stabilisers to modulate PPIs, but also give important insights into the dynamics of ternary complex formation. Finally, we show how native MS can be used as a screening tool to search for PPI stabilisers, highlighting its potential role as a primary screening technology in the hunt for novel therapeutic PPI stabilisers.