RESUMO
Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.
Assuntos
Proteínas de Insetos/metabolismo , Mitocôndrias/enzimologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas Quinases/metabolismo , Tribolium/enzimologia , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Estabilidade Enzimática , Humanos , Proteínas de Insetos/genética , Cinética , Mitocôndrias/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/genética , Simulação de Acoplamento Molecular , Mutação , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Relação Estrutura-Atividade , Tribolium/genéticaRESUMO
Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin-mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin-like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10-fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small-angle X-ray scattering and hydrogen-deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.
Assuntos
Proteínas Quinases/química , Proteínas Quinases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Animais , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Ratos , Serina/química , Serina/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/genética , Ubiquitinação , Domínios de Homologia de srcRESUMO
The enzyme UDP-glucose:glycoprotein glucosyltransferase (UGGT) mediates quality control of glycoproteins in the endoplasmic reticulum by attaching glucose to N-linked glycan of misfolded proteins. As a sensor, UGGT ensures that misfolded proteins are recognized by the lectin chaperones and do not leave the secretory pathway. The structure of UGGT and the mechanism of its selectivity for misfolded proteins have been unknown for 25 years. Here, we used negative-stain electron microscopy and small-angle X-ray scattering to determine the structure of UGGT from Drosophila melanogaster at 18-Å resolution. Three-dimensional reconstructions revealed a cage-like structure with a large central cavity. Particle classification revealed flexibility that precluded determination of a high-resolution structure. Introduction of biotinylation sites into a fungal UGGT expressed in Escherichia coli allowed identification of the catalytic and first thioredoxin-like domains. We also used hydrogen-deuterium exchange mass spectrometry to map the binding site of an accessory protein, Sep15, to the first thioredoxin-like domain. The UGGT structural features identified suggest that the central cavity contains the catalytic site and is lined with hydrophobic surfaces. This enhances the binding of misfolded substrates with exposed hydrophobic residues and excludes folded proteins with hydrophilic surfaces. In conclusion, we have determined the UGGT structure, which enabled us to develop a plausible functional model of the mechanism for UGGT's selectivity for misfolded glycoproteins.
Assuntos
Glucosiltransferases/química , Dobramento de Proteína , Açúcares de Uridina Difosfato/química , Animais , Medição da Troca de Deutério , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Domínios Proteicos , Selenoproteínas/química , Selenoproteínas/genética , Selenoproteínas/metabolismo , Açúcares de Uridina Difosfato/genética , Açúcares de Uridina Difosfato/metabolismoRESUMO
The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.
Assuntos
Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Animais , Sítios de Ligação , Brônquios/citologia , Membrana Celular/metabolismo , Cricetinae , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Glicosilação , Humanos , Mutação , Nucleotídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dobramento de Proteína , Fibrose Cística/genética , Mutação , Retículo Endoplasmático/metabolismoRESUMO
The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.
RESUMO
Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the ß-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.
RESUMO
The human neuraminidases (NEU) consist of a family of four isoforms (NEU1-NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl ß-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (k(rel)). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were substrates, and in the case of the N5-azidoacetyl derivative, the activity was superior to that of GM3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac. We propose a two-site model for enzyme recognition, requiring interaction at both the Neu5Ac residue and the hydrophobic aglycone.
Assuntos
Neuraminidase/metabolismo , Sequência de Carboidratos , Membrana Celular/enzimologia , Análise de Fourier , Glicolipídeos/síntese química , Glicolipídeos/química , Glicolipídeos/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Neuraminidase/química , Espectrometria de Massas por Ionização por Electrospray , Especificidade por SubstratoRESUMO
The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 â 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 â 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.
Assuntos
Galactosiltransferases/genética , Monossacarídeos/metabolismo , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Domínio Catalítico , Galactosiltransferases/química , Galactosiltransferases/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Trissacarídeos/metabolismoRESUMO
A simple method for establishing whether complexes composed of small molecules detected by electrospray ionization mass spectrometry (ES-MS) originate from specific interactions in solution or nonspecific binding during the ES process is described. The technique, referred to as the nonspecific probe method, exploits the tendency of small molecules to bind nonspecifically to macromolecules during the ES process to establish the presence of specific noncovalent interactions. To implement the method, a macromolecule probe (P(NS)), which does not bind specifically to any of the components present in solution, is added prior to ES-MS analysis. The existence of specific small-molecule complexes is determined from an analysis of the measured distributions of the small molecules bound nonspecifically to P(NS). The principal assumption on which this methodology is based is that nonspecific binding of small molecules and their complexes to P(NS) during ES is a statistical (random) process. A mathematical framework for establishing the presence of specific heterocomplexes is presented. The reliability of the method for distinguishing specific from nonspecific small-molecule interactions is illustrated for peptide-antibiotic and metal ion-ligand interactions in water.
Assuntos
Espectrometria de Massas por Ionização por Electrospray/métodos , Sondas MolecularesRESUMO
The first comparative thermodynamic study of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB), interacting with donor substrates, donor and acceptor analogs, and trisaccharide products in vitro is reported. The binding constants, measured at 24 degrees C with the direct electrospray ionization mass spectrometry (ES-MS) assay, provide new insights into these model GTs and their interactions with substrate and product. Notably, the recombinant forms of GTA and GTB used in this study are shown to exist as homodimers, stabilized by noncovalent interactions at neutral pH. In the absence of divalent metal ion, neither GTA nor GTB exhibits any appreciable affinity for its native donors (UDP-GalNAc, UDP-Gal). Upon introduction of Mn(2+), both donors undergo enzyme-catalyzed hydrolysis in the presence of either GTA or GTB. Hydrolysis of UDP-GalNAc in the presence of GTA proceeds very rapidly under the solution conditions investigated and a binding constant could not be directly measured. In contrast, the rate of hydrolysis of UDP-Gal in the presence of GTB is significantly slower and, utilizing a modified approach to analyze the ES-MS data, a binding constant of 2 x 10(4) M(-1) was established. GTA and GTB bind the donor analogs UDP-GlcNAc, UDP-Glc with affinities similar to those measured for UDP-Gal and UDP-GalNAc (GTB only), suggesting that the native donors and donor analogs bind to the GTA and GTB through similar interactions. The binding constant determined for GTA and UDP-GlcNAc (approximately 1 x 10(4) M(-1)), therefore, provides an estimate for the binding constant for GTA and UDP-GalNAc. Binding of GTA and GTB with the A and B trisaccharide products was also investigated for the first time. In the absence of UDP and Mn(2+), both GTA and GTB recognize their respective trisaccharide products but with a low affinity approximately 10(3) M(-1); the presence of UDP and Mn(2+) has no effect on A trisaccharide binding but precludes B-trisaccharide binding.
Assuntos
Sistema ABO de Grupos Sanguíneos/metabolismo , Galactosiltransferases/metabolismo , Oligossacarídeos/metabolismo , Sítios de Ligação , Humanos , Hidrólise , Ligantes , Espectrometria de Massas por Ionização por Electrospray , TermodinâmicaRESUMO
Cystic fibrosis (CF) is one of the most common, lethal autosomal recessive diseases in Caucasians with a life expectancy of 37-47 years. The CF transmembrane conductance regulator (CFTR) is a plasma membrane ion channel, confined to apical membrane of epithelia, and ensures transepithelial water and solute movement across secretory epithelia in several organs. Numerous CF mutations, including the most prevalent deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) leads to CFTR global misfolding and premature intracellular degradation at the endoplasmic reticulum (ER). To better understand the misfolding mechanism caused by CF-causing point mutations in the NBD1, which is poorly understood, differential scanning fluorimetry (DSF) and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) are the choice of techniques. These established methods can measure the conformational dynamics of the NBD1 globally and at peptide resolution level by monitoring backbone amide HDX, respectively, and will be instrumental to evaluate the mechanism of action of CF mutations and folding correctors that rescue CFTR folding defects via stabilizing the mutant NBD1.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Medição da Troca de Deutério/métodos , Fluorometria/métodos , Espectrometria de Massas/métodos , Simulação de Dinâmica Molecular , Mutação Puntual , Sequência de Aminoácidos , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Expressão Gênica , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by in vitro testing of the best virtual hits in recombinant human Caspase-6 activity assays identified novel allosteric Caspase-6 inhibitors with IC50 and Ki values ranging from ~2 to 13 µM. This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.
Assuntos
Caspase 6/química , Caspase 6/metabolismo , Inibidores de Caspase/farmacologia , Mutação de Sentido Incorreto , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Caspase 6/genética , Inibidores de Caspase/química , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Humanos , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Affinities of the human blood group glycosyltransferases, alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) and alpha-(1-->3)-galactosyltransferase (GTB) for their common acceptor substrate alpha-l-Fucp-(1-->2)-beta-d-Galp-O(CH2)(7)CH3 (1), in the absence and presence of bound uridine 5'-diphosphate (UDP) and Mn2+ were determined using temperature-controlled electrospray ionization mass spectrometry. The presence of bound UDP and Mn(2+) in the donor binding site has a marked influence on the thermodynamic parameters for the association of 1 with GTA and GTB. Both the enthalpy and entropy of association (DeltaH(a), DeltaS(a)) decrease significantly. However, the free energy of association (DeltaG(a)) is unchanged at physiological temperature. The differences in the DeltaH(a) and DeltaS(a) values determined in the presence and absence of bound UDP are attributed to structural changes in the glycosyltransferases induced by the simultaneous binding of 1 and UDP.
Assuntos
Antígenos de Grupos Sanguíneos/imunologia , Antígenos de Grupos Sanguíneos/metabolismo , Glicosiltransferases/imunologia , Glicosiltransferases/metabolismo , Temperatura , Antígenos de Grupos Sanguíneos/química , Humanos , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Termodinâmica , Difosfato de Uridina/metabolismoRESUMO
Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson's disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson's disease mutations and the presence of internal linkers that allow large domain movements in parkin.
Assuntos
Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cristalografia por Raios X , Ativação Enzimática , Humanos , Proteínas de Insetos/genética , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Tephritidae/genética , Tephritidae/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
In the version of this article initially published, RING2 in the schematic to the left in Fig. 1b was mislabeled as RING0. The error has been corrected in the HTML and PDF versions of the article.
RESUMO
An investigation of the nonspecific association of small charged biomolecules and proteins in electrospray ionization mass spectrometry (ES-MS) is described. Aqueous solutions containing pairs of proteins and a small acidic or basic biomolecule that does not interact specifically with either of the proteins were analyzed by ES-MS and the distributions of the biomolecules bound nonspecifically to each pair of proteins compared. For the basic amino acid arginine and the peptide RGVFRR, nonequivalent distributions were measured in positive ion mode, but equivalent distributions were measured in negative ion mode. In the case of uridine 5'-diphosphate, nonequivalent distributions were measured in negative ion mode, but equivalent distributions observed in positive ion mode. The results of dissociation experiments performed on the gaseous ions of the nonspecific complexes suggest that the nonequivalent distributions result from differences in the extent to which the nonspecific complexes undergo in-source dissociation. To test this hypothesis, the distributions of nonspecifically bound basic molecules measured in the presence of imidazole, which protects complexes from in-source dissociation, were compared. In all cases, equivalent distributions were obtained. The results indicate that nonspecific binding of charged molecules to proteins during ES is a statistical process, independent of protein structure and size. However, the kinetic stabilities of the nonspecific interactions are sensitive to the nature of the protein ions. It is concluded that the reference protein method for correcting ES mass spectra for nonspecific ligand-protein binding can be applied to the analysis of ionic ligands, provided that in-source dissociation of the nonspecific interactions is minimized.