RESUMO
Phosphatidylinositol (PtdIns) transfer proteins (PITPs) enhance the activities of PtdIns 4-OH kinases that generate signaling pools of PtdIns-4-phosphate. In that capacity, PITPs serve as key regulators of lipid signaling in eukaryotic cells. Although the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activities, the underlying mechanism is not understood. Herein, we apply an integrative structural biology approach to investigate interactions of the yeast PITP Sec14 with small-molecule inhibitors (SMIs) of its phospholipid exchange cycle. Using a combination of X-ray crystallography, solution NMR spectroscopy, and atomistic MD simulations, we dissect how SMIs compete with native Sec14 phospholipid ligands and arrest phospholipid exchange. Moreover, as Sec14 PITPs represent new targets for the development of next-generation antifungal drugs, the structures of Sec14 bound to SMIs of diverse chemotypes reported in this study will provide critical information required for future structure-based design of next-generation lead compounds directed against Sec14 PITPs of virulent fungi.
Assuntos
Antifúngicos , Desenho de Fármacos , Proteínas de Transferência de Fosfolipídeos , Proteínas de Saccharomyces cerevisiae , Transporte Biológico/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Transdução de Sinais , Antifúngicos/química , Antifúngicos/farmacologiaRESUMO
Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl-sn-glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.
Assuntos
Diglicerídeos/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Sítios de Ligação , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca2+ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb2+, a structural and functional surrogate of Ca2+, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb2+-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, "prime" Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.
Assuntos
Domínios C2 , Sinaptotagmina I , Cálcio/metabolismo , Íons , Fosfatidilserinas , Ligação Proteica , Sinaptotagmina I/metabolismo , SinaptotagminasRESUMO
The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T. virens containing a directed mutation in the non-ribosomal peptide synthetase encoding gene Tex7, the appearance of an unknown compound in anomalously large quantities was visualized by TLC. Using a combination of HPLC, LC-MS/MS, and NMR spectroscopy, this compound was identified as heptelidic acid. This discovery alters the strain classification structure of T. virens. Additionally, the Tex7 mutants inhibited growth of maize seedlings, while retaining the ability to induce systemic resistance against the foliar fungal pathogen, Cochliobolus heterostrophus.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Proteínas Fúngicas/genética , Peptídeo Sintases/genética , Trichoderma/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Família Multigênica , Peptídeo Sintases/metabolismo , Sesquiterpenos/metabolismo , Trichoderma/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/microbiologiaRESUMO
Cd2+ and Pb2+ are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd2+ and Pb2+ causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca2+-binding motifs of protein kinase C. The ability of protein oligomers to interact with anionic membranes is enhanced compared to the monomeric species. Our findings suggest that metal-ion-dependent peripheral membrane domains can be utilized for generating protein-metal-ion nanoclusters and serve as biotemplates for the design of sequestration agents.
Assuntos
Cádmio/farmacologia , Membrana Celular/metabolismo , Chumbo/farmacologia , Nanocompostos/química , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Membrana Celular/efeitos dos fármacos , Humanos , Conformação Proteica , Domínios ProteicosRESUMO
Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca2+-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca2+ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.
Assuntos
Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutação , Domínios Proteicos , Proteína Quinase C-alfa/genética , Ratos , Eletricidade EstáticaRESUMO
Conserved homology-1 (C1) domains are peripheral membrane domains that target their host proteins to diacylglycerol (DAG)-containing membranes. It has been previously shown that a conservative aromatic mutation of a single residue in the C1 domain has a profound effect on DAG affinity. We report that the "DAG-toggling" mutation changes the conformational dynamics of the loop region that forms the binding site for the C1 activators. Moreover, there is a correlation among the residue identity at the mutation site, DAG affinity, and loop dynamics in four C1 variants. We propose that "toggling" of DAG affinity may occur through modulation of both protein-membrane interactions and the geometry of the activator-binding cleft, with the loop dynamics being responsible for the latter.
Assuntos
Diglicerídeos/química , Proteína Quinase C/química , Diglicerídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Proteína Quinase C/metabolismo , TermodinâmicaRESUMO
C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.
Assuntos
Cádmio/metabolismo , Membrana Celular/metabolismo , Eletricidade Estática , Sinaptotagmina I/metabolismo , Sítios de Ligação , Cádmio/química , Membrana Celular/química , Cristalografia por Raios X , Humanos , Conformação Proteica , Sinaptotagmina I/químicaRESUMO
Ca2+-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca2+-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca2+ ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca2+ binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.
Assuntos
Cálcio/metabolismo , Proteína Quinase C-alfa/química , Proteína Quinase C-alfa/metabolismo , Regulação Alostérica , Apoenzimas/química , Apoenzimas/metabolismo , Domínios C2 , Ligação de Hidrogênio , Metais/metabolismo , Modelos Moleculares , Ligação ProteicaRESUMO
Protein kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. Significant research efforts have been directed at understanding the structure, function, and regulatory modes of the enzyme since its discovery and identification as the first receptor for tumor-promoting phorbol esters. The activation of PKC involves a transition from the cytosolic autoinhibited latent form to the membrane-associated active form. The membrane recruitment step is accompanied by the conformational rearrangement of the enzyme, which relieves autoinhibitory interactions and thereby allows PKC to phosphorylate its targets. The multidomain structure and intrinsic flexibility of PKC present remarkable challenges and opportunities for the biophysical and structural biology studies of this class of enzymes and their interactions with membranes, the major focus of this Current Topic. I will highlight the recent advances in the field, outline the current challenges, and identify areas where biophysics and structural biology approaches can provide insight into the isoenzyme-specific regulation of PKC activity.
Assuntos
Proteína Quinase C/química , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Fenômenos Biofísicos , Ativação Enzimática , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Biológicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteína Quinase C/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826-830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities.
Assuntos
Diglicerídeos/metabolismo , Proteína Quinase C-delta/química , Proteína Quinase C-delta/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Diglicerídeos/química , Cinética , Modelos Moleculares , Ligação Proteica , Proteína Quinase C-delta/genética , Estrutura Terciária de Proteína , RatosRESUMO
We report the protocol for heterologous expression and purification of the N-terminal regulatory region of two Protein Kinase C (PKC)(1) isozymes, one conventional and one novel. Previous studies of these domains relied almost exclusively on the fusion constructs with high-molecular-weight solubility fusion partners such as GST and MBP. We developed experimental procedures that enabled us to overcome challenges associated with the amphiphilic character of the regulatory domain and generate sufficient quantities of fusion partner-free proteins for biophysical work. The key features of the protocol are the identity of the cleavable fusion partner, expression conditions, growth medium additives, introduction of mutation/solubility tags, and incorporation of osmolytes. The protein yields are sufficient for cost-effective production of isotopically enriched proteins for NMR work and biophysical studies in general. Our work opens up an avenue for the structural studies of these challenging proteins with high amphiphilic character.
Assuntos
Proteína Quinase C-alfa/isolamento & purificação , Proteína Quinase C-delta/isolamento & purificação , Proteína Quinase C/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Concentração Osmolar , Plasmídeos/química , Plasmídeos/metabolismo , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteína Quinase C-alfa/biossíntese , Proteína Quinase C-alfa/genética , Proteína Quinase C-delta/biossíntese , Proteína Quinase C-delta/genética , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de SequênciaRESUMO
One of the mechanisms by which toxic metal ions interfere with cellular functions is ionic mimicry, where they bind to protein sites in lieu of native metals Ca 2+ and Zn 2+ . The influence of crowded intracellular environments on these interactions is not well understood. Here, we demonstrate the application of in-cell and lysate NMR spectroscopy to obtain atomic-level information on how a potent environmental toxin cadmium interacts with its protein targets. The experiments, conducted in intact E. coli cells and their lysates, revealed that Cd 2+ can profoundly affect the quinary interactions of its protein partners, and can replace Zn 2+ in both labile and non-labile protein structural sites without significant perturbation of the membrane binding function. Surprisingly, in crowded molecular environments Cd 2+ can effectively target not only all-sulfur and mixed sulfur/nitrogen but also all-oxygen coordination sites. The sulfur-rich coordination environments show significant promise for bioremedial applications, as demonstrated by the ability of the designed protein scaffold α 3 DIV to sequester intracellular cadmium. Our data suggests that in-cell NMR spectroscopy is a powerful tool for probing interactions of toxic metal ions with their potential protein targets, and for the assessment of potency of sequestering agents.
RESUMO
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Ligação Proteica , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Humanos , Proteína Quinase C/metabolismo , Proteína Quinase C/química , Proteína Quinase C/genética , Conformação ProteicaRESUMO
Due to its favorable spectroscopic properties, Cd(2+) is frequently used as a probe of Ca(2+) sites in proteins. We investigate the ability of Cd(2+) to act as a structural and functional surrogate of Ca(2+) in protein-membrane interactions. C2 domain from protein kinase Cα (C2α) was chosen as a paradigm for the Ca(2+)-dependent phosphatidylserine-binding peripheral membrane domains. We identified the Cd(2+)-binding sites of C2α using NMR spectroscopy, determined the 1.6 Å crystal structure of Cd(2+)-bound C2α, and characterized metal-ion-dependent interactions between C2α and phospholipid membranes using fluorescence spectroscopy and ultracentrifugation experiments. We show that Cd(2+) forms a tight complex with the membrane-binding loops of C2α but is unable to support its membrane-binding function. This is in sharp contrast with Pb(2+), which is almost as effective as Ca(2+) in driving the C2α-membrane association process. Our results provide the first direct evidence for the specific role of divalent metal ions in mediating protein-membrane interactions, have important implications for metal substitution studies in proteins, and illustrate the potential diversity of functional responses caused by toxic metal ions.
Assuntos
Cádmio/química , Cálcio/química , Fosfolipídeos/química , Proteína Quinase C-alfa/química , Modelos Moleculares , Proteína Quinase C-alfa/metabolismoRESUMO
The peptidyl-prolyl isomerase Pin1 cooperates with proline-directed kinases and phosphatases to regulate multiple oncogenic pathways. Pin1 specifically recognizes phosphorylated Ser/Thr-Pro motifs in proteins and catalyzes their cis-trans isomerization. The Pin1-catalyzed conformational changes determine the stability, activity, and subcellular localization of numerous protein substrates. We conducted a survey of eukaryotic protein kinases that are regulated by Pin1 and whose Pin1 binding sites have been identified. Our analyses reveal that Pin1 target sites in kinases do not fall exclusively within the intrinsically disordered regions of these enzymes. Rather, they fall into three groups based on their location: (i) within the catalytic kinase domain, (ii) in the C-terminal kinase region, and (iii) in regulatory domains. Some of the kinases downregulated by Pin1 activity are tumor-suppressing, and all kinases upregulated by Pin1 activity are functionally pro-oncogenic. These findings further reinforce the rationale for developing Pin1-specific inhibitors as attractive pharmaceuticals for cancer therapy.
Assuntos
Neoplasias , Peptidilprolil Isomerase , Humanos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Eucariotos/metabolismo , Proteínas Quinases/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a compact conformation in which it engages two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, the latter being a non-canonical Pin1-interacting element. The structural information, combined with the results of extensive binding studies and in vivo experiments suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
RESUMO
Ca(2+)-responsive C2 domains are peripheral membrane modules that target their host proteins to anionic membranes upon binding Ca(2+) ions. Several C2 domain-containing proteins, such as protein kinase C isoenzymes (PKCs), have been identified as molecular targets of Pb(2+), a known environmental toxin. We demonstrated previously that the C2 domain from PKCα (C2α) binds Pb(2+) with high affinity and undergoes membrane insertion in the Pb(2+)-complexed form. The objective of this work was to determine the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) on the C2α-Pb(2+) interactions. Using nuclear magnetic resonance (NMR) experiments, we show that Pb(2+) and PIP(2) synergistically enhance each other's affinity for C2α. Moreover, the affinity of C2α for PIP(2) increases upon progressive saturation of the metal-binding sites. Combining the NMR data with the results of protein-to-membrane Förster resonance energy transfer and vesicle sedimentation experiments, we demonstrate that PIP(2) can influence two aspects of C2α-Pb(2+)-membrane interactions: the affinity of C2α for Pb(2+) and the association of Pb(2+) with the anionic sites on the membrane. Both factors may contribute to the toxic effect of Pb(2+) resulting from the aberrant modulation of PKCα activity. Finally, we propose a mechanism for Pb(2+) outcompeting Ca(2+) from membrane-bound C2α.
Assuntos
Membrana Celular/metabolismo , Chumbo/química , Fosfatidilinositol 4,5-Difosfato/química , Sítios de Ligação , Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Chumbo/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C/metabolismo , Estrutura Terciária de ProteínaRESUMO
Structural cysteine-rich Zn(2+) sites that stabilize protein folds are considered to be unreactive. In this article, we identified a reactive cysteine residue, Cys151, in a treble-clef zinc finger with a Cys(3)His coordination sphere. The protein in question is the C1B domain of Protein Kinase Cα (PKCα). Mass-tagging cysteine assays of several C1B variants were employed to ascertain the site specificity of the covalent modification. The reactivity of Cys151 in C1B also manifests itself in the structural dynamics of the Zn(2+) coordination sphere where the Sγ of Cys151 alternates between the Zn(2+)-bound thiolate and free thiol states. We used NMR-detected pH titrations, ZZ-exchange spectroscopy, and residual dipolar coupling (RDC)-driven structure refinement to characterize the two exchanging conformations of C1B that differ in zinc coordination. Our data suggest that Cys151 serves as an entry point for the reactive oxygen species that activate PKCα in a process involving Zn(2+) release.
Assuntos
Cisteína/química , Proteína Quinase C-alfa/química , Espécies Reativas de Oxigênio/química , Zinco/química , Animais , Sítios de Ligação , Cisteína/metabolismo , Camundongos , Ressonância Magnética Nuclear Biomolecular , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/metabolismo , Zinco/metabolismo , Dedos de ZincoRESUMO
Virulent fungi represent a particularly difficult problem in the infectious disease arena as these organisms are eukaryotes that share many orthologous activities with their human hosts. The fact that these activities are often catalyzed by conserved proteins places additional demands on development of pharmacological strategies for specifically inhibiting target fungal activities without imposing undesirable secondary effects on the host. While deployment of a limited set of anti-mycotics has to date satisfied the clinical needs for treatment of fungal infections, the recent emergence of multi-drug resistant fungal 'superbugs' now poses a serious global health threat with rapidly diminishing options for treatment. This escalating infectious disease problem emphasizes the urgent need for development of new classes of anti-mycotics. In that regard, Sec14 phosphatidylinositol transfer proteins offer interesting possibilities for interfering with fungal phosphoinositide signaling with exquisite specificity and without targeting the highly conserved lipid kinases responsible for phosphoinositide production. Herein, we review the establishment of proof-of-principle that demonstrates the feasibility of such an approach. We also describe the lead compounds of four chemotypes that directly target fungal Sec14 proteins. The rules that pertain to the mechanism(s) of Sec14 inhibition by validated small molecule inhibitors, and the open questions that remain, are discussed - as are the challenges that face development of next generation Sec14-directed inhibitors.