ABSTRACT
Combination of biophysical and structural techniques allowed characterizing and uncovering the mechanisms underlying increased binding affinity of lactosamine derivatives for galectin 3. In particular, complementing information gathered from X-ray crystallography, native mass spectrometry and isothermal microcalorimetry showed favorable enthalpic contribution of cation-π interaction between lactosamine aryl substitutions and arginine residues from the carbohydrate recognition domain, which resulted in two log increase in compound binding affinity. This incrementing strategy allowed individual contribution of galectin inhibitor moieties to be dissected. Altogether, our results suggest that core and substituents of these saccharide-based inhibitors can be optimized separately, providing valuable tools to study the role of galectins in diseases.
Subject(s)
Amino Sugars/chemistry , Amino Sugars/pharmacology , Galectin 3/metabolism , Blood Proteins , Calorimetry , Crystallography, X-Ray , Galectin 3/biosynthesis , Galectin 3/chemistry , Galectin 3/isolation & purification , Galectins , Humans , Mass Spectrometry , Models, Molecular , Molecular Conformation , Structure-Activity RelationshipABSTRACT
Fragment-based lead discovery is gaining momentum in drug development. Typically, a hierarchical cascade of several screening techniques is consulted to identify fragment hits which are then analyzed by crystallography. Because crystal structures with bound fragments are essential for the subsequent hit-to-lead-to-drug optimization, the screening process should distinguish reliably between binders and non-binders. We therefore investigated whether different screening methods would reveal similar collections of putative binders. First we used a biochemical assay to identify fragments that bind to endothiapepsin, a surrogate for disease-relevant aspartic proteases. In a comprehensive screening approach, we then evaluated our 361-entry library by using a reporter-displacement assay, saturation-transfer difference NMR, native mass spectrometry, thermophoresis, and a thermal shift assay. While the combined results of these screening methods retrieve 10 of the 11 crystal structures originally predicted by the biochemical assay, the mutual overlap of individual hit lists is surprisingly low, highlighting that each technique operates on different biophysical principles and conditions.
Subject(s)
Biochemistry/methods , Biophysics/methods , High-Throughput Screening Assays/methods , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Drug Discovery/methods , Magnetic Resonance Spectroscopy , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
A 7SKsnRNP complex, comprising the non-coding RNA 7SK and proteins MePCE and LARP7, participates in the regulation of the transcription elongation by RNA-polymerase II in higher eukaryotes. Binding of a HEXIM protein triggers the inhibition of the kinase complex P-TEFb, a key actor of the switch from paused transcription to elongation. The present paper reviews what is known about the specific recognition of the 7SK RNA by the HEXIM protein. HEXIM uses an arginine-rich motif (ARM) peptide to bind one specific site in the 5'-hairpin of the 7SK RNA. Since HEXIM forms a dimer, what happens with the second ARM impacts the assembly symmetry. In order to help sort through possible models, a combination of native mass spectrometry and electrophoretic mobility shift assays was used. It provides evidence that only one ARM of the HEXIM dimer is directly binding to the RNA hairpin and that another sequence downstream of the ARM participates in a second binding event allowing the other monomer of HEXIM to bind the RNA.
Subject(s)
Methyltransferases/metabolism , RNA, Untranslated/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoproteins/metabolism , Binding Sites/genetics , Electrophoretic Mobility Shift Assay , Humans , Mass Spectrometry , Methyltransferases/chemistry , Methyltransferases/genetics , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Protein Structure, Tertiary , RNA, Untranslated/chemistry , RNA, Untranslated/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribonucleoproteins/chemistry , Ribonucleoproteins/genetics , Transcription FactorsABSTRACT
Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Mass Spectrometry/methods , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/metabolism , Archaeal Proteins/analysis , Ribonucleoproteins, Small Nuclear/analysis , Systems BiologyABSTRACT
Keap1 binds to the Nrf2 transcription factor to promote its degradation, resulting in the loss of gene products that protect against oxidative stress. While cell-active small molecules have been identified that modify cysteines in Keap1 and effect the Nrf2 dependent pathway, few act through a non-covalent mechanism. We have identified and characterized several small molecule compounds that specifically bind to the Keap1 Kelch-DC domain as measured by NMR, native mass spectrometry and X-ray crystallography. One compound upregulates Nrf2 response genes measured by a luciferase cell reporter assay. The non-covalent inhibition strategy presents a reasonable course of action to avoid toxic side-effects due to non-specific cysteine modification.
Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , NF-E2-Related Factor 2/antagonists & inhibitors , NF-E2-Related Factor 2/metabolism , Small Molecule Libraries/pharmacology , Carrier Proteins , Crystallography, X-Ray , Intracellular Signaling Peptides and Proteins/chemistry , Kelch-Like ECH-Associated Protein 1 , NF-E2-Related Factor 2/chemistry , Protein Binding/drug effects , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Structure-Activity Relationship , ThermodynamicsABSTRACT
Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need therefore to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their posttranslational modifications with the aim of reducing their chemistry, manufacturing, and control liabilities. This chapter will provide the reader with a description of the general approach allowing antibody/antigen systems to be characterized by noncovalent MS. In the present chapter, we describe how recent noncovalent MS technologies are used to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) over-expressed in tumor cells. We will detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of noncovalent antibody/antigen complexes by MS. We will then focus on the type and the reliability of the information that we get from noncovalent MS data, with emphasis on the determination of the stoichiometry of antibody/antigen systems. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , Immunoglobulin G/chemistry , Animals , Buffers , CHO Cells , Cricetinae , Humans , Hybridomas , Spectrometry, Mass, Electrospray Ionization/methodsABSTRACT
PVL (Panton-Valentine leukocidin) and other Staphylococcus aureus ß-stranded pore-forming toxins are important virulence factors involved in various pathologies that are often necrotizing. The present study characterized leukotoxin inhibition by selected SCns (p-sulfonato-calix[n]arenes): SC4, SC6 and SC8. These chemicals have no toxic effects on human erythrocytes or neutrophils, and some are able to inhibit both the activity of and the cell lysis by leukotoxins in a dose-dependent manner. Depending on the type of leukotoxins and SCns, flow cytometry revealed IC50 values of 6-22 µM for Ca2+ activation and of 2-50 µM for cell lysis. SCns were observed to affect membrane binding of class S proteins responsible for cell specificity. Electrospray MS and surface plasmon resonance established supramolecular interactions (1:1 stoichiometry) between SCns and class S proteins in solution, but not class F proteins. The membrane-binding affinity of S proteins was Kd=0.07-6.2 nM. The binding ability was completely abolished by SCns at different concentrations according to the number of benzenes (30-300 µM; SC8>SC6â«SC4). The inhibitory properties of SCns were also observed in vivo in a rabbit model of PVL-induced endophthalmitis. These calixarenes may represent new therapeutic avenues aimed at minimizing inflammatory reactions and necrosis due to certain virulence factors.
Subject(s)
Calixarenes/pharmacology , Exotoxins/antagonists & inhibitors , Exotoxins/metabolism , Staphylococcus aureus/metabolism , Animals , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/metabolism , Calixarenes/metabolism , Down-Regulation/drug effects , Drug Evaluation, Preclinical , Hemolysin Proteins/antagonists & inhibitors , Hemolysin Proteins/metabolism , Humans , Macromolecular Substances/metabolism , Models, Biological , Phenols/metabolism , Phenols/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Rabbits , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/metabolism , Staphylococcus aureus/pathogenicity , Virulence Factors/antagonists & inhibitors , Virulence Factors/metabolismABSTRACT
The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.
Subject(s)
Aldehyde Reductase/chemistry , Aldehyde Reductase/metabolism , Aldehyde Reductase/antagonists & inhibitors , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Ligands , Models, Molecular , Protein BindingABSTRACT
Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.
Subject(s)
Ions/analysis , Mass Spectrometry/methods , bcl-X Protein/analysis , Amino Acid Sequence , Biphenyl Compounds/chemistry , Crystallography, X-Ray , Helium , Humans , Ligands , Mass Spectrometry/instrumentation , Molecular Sequence Data , Nitrogen , Nitrophenols/chemistry , Piperazines/chemistry , Protein Conformation , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Sensitivity and Specificity , Sequence Alignment , Sulfonamides/chemistry , bcl-X Protein/chemistryABSTRACT
The human USP7 deubiquitinating enzyme was shown to regulate many proteins involved in the cell cycle, as well as tumor suppressors and oncogenes. Thus, USP7 offers a promising, strategic target for cancer therapy. Using biochemical assays and activity-based protein profiling in living systems, we identified small-molecule antagonists of USP7 and demonstrated USP7 inhibitor occupancy and selectivity in cancer cell lines. These compounds bind USP7 in the active site through a covalent mechanism. In cancer cells, these active-site-targeting inhibitors were shown to regulate the level of several USP7 substrates and thus recapitulated the USP7 knockdown phenotype that leads to G1 arrest in colon cancer cells. The data presented in this report provide proof of principle that USP7 inhibitors may be a valuable therapeutic for cancer. In addition, the discovery of such molecules offers interesting tools for studying deubiquitination.
Subject(s)
Enzyme Inhibitors/chemistry , Ubiquitin Thiolesterase/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , G1 Phase Cell Cycle Checkpoints/drug effects , HCT116 Cells , Humans , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Specific Peptidase 7ABSTRACT
Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using (14)N versus (15)N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.
Subject(s)
Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Substitution , Crystallography, X-Ray , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Glutamine/chemistry , Glutamine/metabolism , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Nitrogen Isotopes , Protein MultimerizationABSTRACT
BACKGROUND: Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. METHODOLOGY/PRINCIPAL FINDINGS: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. CONCLUSIONS/SIGNIFICANCE: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Catalytic Domain , Protein Multimerization , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Binding Sites/genetics , Binding, Competitive , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Death-Associated Protein Kinases , Fluorescence Polarization , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Humans , Molecular Structure , Mutation , Peptides/chemistry , Peptides/metabolism , Protein Binding , Spectrometry, Mass, Electrospray Ionization , Substrate SpecificityABSTRACT
The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, ï¬uorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members.
Subject(s)
DNA, Bacterial/chemistry , Repressor Proteins/chemistry , Carbohydrates/chemistry , Chromatography, Gel , Mass Spectrometry , Models, Molecular , Operator Regions, Genetic , Scattering, Small Angle , Spectrometry, Fluorescence , X-Ray DiffractionABSTRACT
The central glycolytic genes repressor (CggR) is a 37 kDa transcriptional repressor protein which plays a key role in Bacillus subtilis glycolysis by regulating the transcription of the gapA operon. Fructose-1,6-bisphosphate (FBP), identified as the effector sugar, has been shown to abolish the binding cooperativity of CggR to its DNA target and to modify the conformational dynamics of the CggR/DNA complex. In the present study, noncovalent mass spectrometry (MS) was used to obtain deeper insights into FBP-dependent CggR/DNA interactions. The effect of FBP binding on CggR alone and on CggR/DNA complexes was examined using automated chip-based nanoelectrospray MS and traveling wave ion mobility mass spectrometry (IM-MS). Our results revealed that tetrameric CggR dissociates into dimers upon FBP binding. Moreover, FBP binding to CggR/DNA complexes triggers disruption of intermolecular protein/protein interactions within the complex, significantly modifying its conformation as evidenced by a 5% increase of its collision cross section. For the first time, the use of IM-MS is reported to probe ligand-induced conformational modifications of a protein/DNA complex with an emphasis on the comparison with solution-based techniques.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/chemistry , DNA-Binding Proteins , Fructosediphosphates/pharmacology , Mass Spectrometry , Repressor Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization , Bacillus subtilis/enzymology , DNA-Binding Proteins/drug effects , Gene Expression Regulation, Bacterial , Glycolysis , Protein Conformation/drug effectsABSTRACT
Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases including cancers, immunological disorders, and other pathologies. These large biomolecules display specific structural features, which affect their efficiency and need, therefore, to be extensively characterized using sensitive and orthogonal analytical techniques. Among them, mass spectrometry (MS) has become the method of choice to study mAb amino acid sequences as well as their post-translational modifications. In the present work, recent noncovalent MS-technologies including automated chip-based nanoelectrospray MS and traveling wave ion mobility MS were used for the first time to characterize immune complexes involving both murine and humanized mAb 6F4 directed against human JAM-A, a newly identified antigenic protein (Ag) overexpressed in tumor cells. MS-based structural insights evidenced that heterogeneous disulfide bridge pairings of recombinant JAM-A alter neither its native structure nor mAbs 6F4 recognition properties. Investigations focused on mAb:Ag complexes revealed that, similarly to murine mAb, humanized mAb 6F4 binds selectively up to four antigen molecules with a similar affinity, confirming in this way the reliability of the humanization process. Noncovalent MS appears as an additional supporting technique for therapeutic mAbs lead characterization and development.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigen-Antibody Complex/chemistry , Disulfides/chemistry , Immunoglobulins/chemistry , Pharmaceutical Preparations/chemistry , Recombinant Proteins/chemistry , Spectrometry, Mass, Electrospray Ionization , Animals , Antibodies, Monoclonal/isolation & purification , Cell Adhesion Molecules/immunology , Humans , Immunoglobulins/immunology , Mice , Peptide Fragments/chemistry , Protein Binding , Receptors, Cell SurfaceABSTRACT
The tRNA-modifying enzyme tRNA-guanine transglycosylase (Tgt) is a putative target for new selective antibiotics against Shigella bacteria. The formation of a Tgt homodimer was suggested on the basis of several crystal structures of Tgt in complex with RNA. In the present study, noncovalent mass spectrometry was used (i) to confirm the dimeric oligomerization state of Tgt in solution and (ii) to evidence the binding stoichiometry of the complex formed between Tgt and its full-length substrate tRNA. To further investigate the importance of Tgt protein-protein interaction, point mutations were introduced into the dimer interface in order to study their influence on the formation of the catalytically active complex. Enzyme kinetics revealed a reduced catalytic activity of these mutated variants, which could be related to a destabilization of the dimer formation as evidenced by both noncovalent mass spectrometry and X-ray crystallography. Finally, the effect of inhibitor binding was investigated by noncovalent mass spectrometry, thus providing the binding stoichiometries of Tgt:inhibitor complexes and showing competitive interactions in the presence of tRNA. Inhibitors that display an influence on the formation of the dimer interface in the crystal structure are promising candidates to alter the protein-protein interaction, which could provide a new way to inhibit Tgt.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Pentosyltransferases/chemistry , Pentosyltransferases/metabolism , Protein Conformation , RNA, Transfer , Bacterial Proteins/genetics , Crystallography, X-Ray/methods , Guanine/analogs & derivatives , Guanine/chemistry , Isomerases/chemistry , Isomerases/metabolism , Mass Spectrometry/methods , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Pentosyltransferases/genetics , Point Mutation , Protein Multimerization , RNA, Transfer/chemistry , RNA, Transfer/metabolismABSTRACT
BACKGROUND: The phosphatidylethanolamine-binding protein (PEBP/RKIP), initially found to bind phosphatidylethanolamine (PE), has been shown to be associated with morphine derivatives. Our recent study on bovine primary chromaffin cells showed that inside secretory granules, PEBP is noncovalently associated to endogenous morphine-6-glucuronide (M6G), a highly analgesic morphine metabolite. During stress, M6G-PEBP complexes may be released into circulation to target peripheral opioid receptors. We now report the investigation of PEBP binding properties towards morphine and morphine analogs. MATERIAL/METHODS: Noncovalent electrospray ionization mass spectrometry (ESI-MS) was used to investigate bovine and human PEBP binding properties towards morphine and morphine-glucuronides. RESULTS: We describe for the first time that: (i) PEBP directly interacts with morphine glucuronides (M3G and M6G) but not with morphine, (ii) that the presence of a glucuronide group either on the 3rd or the 6th morphine's carbon does not affect these interactions, (iii) that M6G binds PEBP in a similar manner as the reference ligand PE and (iv) that PEBP displays a similar affinity for PE, M6G and M3G. CONCLUSIONS: Our results suggest that PEBP might protect M6G following its secretion into blood, leading to a longer half life. This study highlights the potentialities of ESI-MS to validate / invalidate the formation of protein: ligand noncovalent complexes when low affinity binders (i.e., compounds with affinities lower than 10(3) M(-1)) are concerned.
Subject(s)
Mass Spectrometry , Morphine Derivatives/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Animals , Cattle , Humans , Morphine Derivatives/chemistry , Phosphatidylethanolamines/metabolism , TitrimetryABSTRACT
The predilection of the beta-crystallin B2 subunit to interact with the betaB3 subunit rather than self associate is evident by the detection of the betaB2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47,450 +/- 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the betaB2 subunit and six of the betaB3 subunit were found to oxidise, with far greater oxidation observed within the betaB3 versus the betaB2 subunit. This, and the observation that the oxidation data of betaB2 subunit is inconsistent with the structure of the betaB2 monomer, demonstrates that the protection of betaB2 is conferred by its association with betaB3 subunit within the heterodimer where only the residues of, and towards, its N-terminal domain remain exposed to solvent. The results suggest that the betaB2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the betaB3 subunit within the heterodimer.
Subject(s)
Mass Spectrometry/methods , beta-Crystallin B Chain/chemistry , Amino Acid Sequence , Animals , Cattle , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Oxidation-Reduction , Peptide Fragments/chemistry , Protein Conformation , Protein Multimerization , Trypsin/metabolism , beta-Crystallin B Chain/metabolismABSTRACT
In the present chapter we detail how mass spectrometry (MS) can be used to characterize noncovalent complexes, especially multimeric proteins and protein/ligand complexes. This original application of MS, also called "supramolecular MS" or "nondenaturing MS," appeared in the early 1990s and has continuously evolved since then. Nondenaturing MS is now fully integrated in structural biology programs and in drug discovery platforms. Indeed, appropriate sample preparation and fine tuning of the instrument make it possible to transfer weak assemblies without disruption from solution into the gas phase of the mass spectrometer. In this chapter we detail experimental conditions (sample preparation, optimization of instrumental parameters, etc.) required for the detection of noncovalent complexes by MS. We then focus on the type of information and accuracy that we get after interpreting electrospray ionization mass spectra obtained under nondenaturing conditions, with emphasis on the determination of the stoichiometry of protein/protein and protein/ligand complexes.
Subject(s)
Mass Spectrometry/methods , Multiprotein Complexes/chemistry , Aldehyde Reductase/antagonists & inhibitors , Aldehyde Reductase/chemistry , Bacterial Proteins/analysis , Ligands , Protein Binding , Protein Serine-Threonine Kinases/analysis , Reproducibility of ResultsABSTRACT
Methyltransferases from the m(1)A(58) tRNA methyltransferase (TrmI) family catalyze the S-adenosyl-l-methionine-dependent N(1)-methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus TrmI, in complex with S-adenosyl-l-homocysteine, was determined at 1.7 A resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI, and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis, and molecular docking. First, we confirmed the tetrameric oligomerization state of TrmI, and we showed that this protein remains tetrameric upon tRNA binding, with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Second, three key residues for the methylation reaction were identified: the universally conserved D170 and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N(9)-methyladenine in the active site of TrmI. The N(9)-methyladenine snugly fits into the catalytic cleft, where the side chain of D170 acts as a bidentate ligand binding the amino moiety of S-adenosyl-l-methionine and the exocyclic amino group of the adenosine. Y194 interacts with the N(9)-methyladenine ring, whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78, and Y194) are essential for fashioning an optimized shape of the catalytic pocket.