RESUMO
Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.
Assuntos
Evolução Molecular , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Aurora Quinase A/química , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Domínio Catalítico , Sequência Conservada , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphA3 , Relação Estrutura-AtividadeRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1005885.].
RESUMO
The catalytic activation of protein kinases requires precise positioning of key conserved catalytic and regulatory motifs in the kinase core. The Regulatory Spine (RS) is one such structural motif that is dynamically assembled upon kinase activation. The RS is also a mutational hotspot in cancers; however, the mechanisms by which cancer mutations impact RS assembly and kinase activity are not fully understood. In this study, through mutational analysis of patient derived mutations in the RS of EGFR kinase, we identify an activating mutation, M766T, at the RS3 position. RS3 is located in the regulatory αC-helix, and a series of mutations at the RS3 position suggest a strong correlation between the amino acid type present at the RS3 position and ligand (EGF) independent EGFR activation. Small polar amino acids increase ligand independent activity, while large aromatic amino acids decrease kinase activity. M766T relies on the canonical asymmetric dimer for full activation. Molecular modeling and molecular dynamics simulations of WT and mutant EGFR suggest a model in which M766T activates the kinase domain by disrupting conserved autoinhibitory interactions between M766 and hydrophobic residues in the activation segment. In addition, a water mediated hydrogen bond network between T766, the conserved K745-E762 salt bridge, and the backbone amide of the DFG motif is identified as a key determinant of M766T-mediated activation. M766T is resistant to FDA approved EGFR inhibitors such as gefitinib and erlotinib, and computational estimation of ligand binding free energy identifies key residues associated with drug sensitivity. In sum, our studies suggest an unusual mode of RS assembly and oncogenic EGFR activation, and provide new clues for the design of allosteric protein kinase inhibitors.
Assuntos
Motivos de Aminoácidos , Domínio Catalítico , Receptores ErbB/genética , Mutação , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Células CHO , Cricetinae , Cricetulus , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/química , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/farmacologia , Gefitinibe , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Immunoblotting , Lapatinib , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Quinazolinas/farmacologiaRESUMO
Protein kinases represent a large and diverse family of evolutionarily related proteins that are abnormally regulated in human cancers. Although genome sequencing studies have revealed thousands of variants in protein kinases, translating "big" genomic data into biological knowledge remains a challenge. Here, we describe an ontological framework for integrating and conceptualizing diverse forms of information related to kinase activation and regulatory mechanisms in a machine readable, human understandable form. We demonstrate the utility of this framework in analyzing the cancer kinome, and in generating testable hypotheses for experimental studies. Through the iterative process of aggregate ontology querying, hypothesis generation and experimental validation, we identify a novel mutational hotspot in the αC-ß4 loop of the kinase domain and demonstrate the functional impact of the identified variants in epidermal growth factor receptor (EGFR) constitutive activity and inhibitor sensitivity. We provide a unified resource for the kinase and cancer community, ProKinO, housed at http://vulcan.cs.uga.edu/prokino.
Assuntos
Neoplasias/enzimologia , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Mineração de Dados , Gefitinibe , Ontologia Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bases de Conhecimento , Modelos Moleculares , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Quinazolinas/farmacologia , Alinhamento de Sequência , SoftwareRESUMO
Cancer is a genetic disease that develops through a series of somatic mutations, a subset of which drive cancer progression. Although cancer genome sequencing studies are beginning to reveal the mutational patterns of genes in various cancers, identifying the small subset of "causative" mutations from the large subset of "non-causative" mutations, which accumulate as a consequence of the disease, is a challenge. In this article, we present an effective machine learning approach for identifying cancer-associated mutations in human protein kinases, a class of signaling proteins known to be frequently mutated in human cancers. We evaluate the performance of 11 well known supervised learners and show that a multiple-classifier approach, which combines the performances of individual learners, significantly improves the classification of known cancer-associated mutations. We introduce several novel features related specifically to structural and functional characteristics of protein kinases and find that the level of conservation of the mutated residue at specific evolutionary depths is an important predictor of oncogenic effect. We consolidate the novel features and the multiple-classifier approach to prioritize and experimentally test a set of rare unconfirmed mutations in the epidermal growth factor receptor tyrosine kinase (EGFR). Our studies identify T725M and L861R as rare cancer-associated mutations inasmuch as these mutations increase EGFR activity in the absence of the activating EGF ligand in cell-based assays.
Assuntos
Mutação , Neoplasias/enzimologia , Oncogenes , Proteínas Quinases/metabolismo , Inteligência Artificial , Humanos , Proteínas Quinases/genéticaRESUMO
Here, we show the binding results of a leguminosae lectin, winged bean basic agglutinin (WBA I) to N-trifluoroacetylgalactosamine (NTFAGalN), methyl-α-N-trifluoroacetylgalactosamine (MeαNTFAGalN) and methyl-ß-tifluoroacetylgalactosamine (MeßNTFAGalN) using (19) F NMR spectroscopy. No chemical shift difference between the free and bound states for NTFAGalN and MeßNTFAGalN, and 0.01-ppm chemical shift change for MeαNTFAGalN, demonstrate that the MeαNTFAGalN has a sufficiently long residence time on the protein binding site as compared to MeßNTFAGalN and the free anomers of NTFAGalN. The sugar anomers were found in slow exchange with the binding site of agglutinin. Consequently, we obtained their binding parameters to the protein using line shape analyses. Aforementioned analyses of the activation parameters for the interactions of these saccharides indicate that the binding of α and ß anomers of NTFAGalN and MeαNTFAGalN is controlled enthalpically, while that of MeßNTFAGalN is controlled entropically. This asserts the sterically constrained nature of the interaction of the MeßNTFAGalN with WBA I. These studies thus highlight a significant role of the conformation of the monosaccharide ligands for their recognition by WBA I.
Assuntos
Acetilglucosamina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Lectinas de Plantas/químicaRESUMO
Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the tree of life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3Ks and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. By identifying a potential link between FN3Ks, redox regulation, and NAD-dependent metabolic processes, our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance is altered.
RESUMO
Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the Tree of Life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) HepG2 cell line alongside an integrative multi-omics study combining transcriptomics, metabolomics, and interactomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), and carbon and co-factor metabolism. Moreover, enrichment of nicotinamide adenine dinucleotide (NAD) binding proteins and localization of human FN3K (HsFN3K) to mitochondria suggests potential links between FN3K and NAD-mediated energy metabolism and redox balance. We report specific binding of HsFN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. Our studies provide a framework for targeting these understudied kinases in diabetic complications and metabolic disorders where redox balance and NAD-dependent metabolic processes are altered.
Assuntos
Redes e Vias Metabólicas , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Células Hep G2 , Redes e Vias Metabólicas/genética , Metabolômica/métodos , NAD/metabolismo , Estresse Oxidativo/fisiologia , Estresse Oxidativo/genética , MultiômicaRESUMO
In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.
RESUMO
Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory 'tail' segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to 'supercharge' the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a 'Swiss-army' assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.
RESUMO
Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The doublecortin-like kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory 'tail' segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations, and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to 'supercharge' the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other calcium calmodulin kinases (CAMKs), and a 'Swiss Army' assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for autoregulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically divergent DCLK1 modulators, stabilizers, or degraders.
Assuntos
Evolução Biológica , Proteínas Serina-Treonina Quinases , Humanos , Isoformas de Proteínas/genética , Proteínas Serina-Treonina Quinases/genética , Processamento Alternativo , Cálcio da Dieta , Quinases Semelhantes a DuplacortinaRESUMO
The cellular response to hypoxia involves several signalling pathways that mediate adaptation and survival. REDD1 (regulated in development and DNA damage responses 1), a hypoxia-inducible factor-1 target gene, has a crucial role in inhibiting mammalian target of rapamycin complex 1 (mTORC1) signalling during hypoxic stress. However, little is known about the signalling pathways and post-translational modifications that regulate REDD1 function. Here, we show that REDD1 is subject to ubiquitin-mediated degradation mediated by the CUL4A-DDB1-ROC1-beta-TRCP E3 ligase complex and through the activity of glycogen synthase kinase 3beta. Furthermore, REDD1 degradation is crucially required for the restoration of mTOR signalling as cells recover from hypoxic stress. Our findings define a mechanism underlying REDD1 degradation and its importance for regulating mTOR signalling.
Assuntos
Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Quinases/metabolismo , Fatores de Transcrição/fisiologia , Proteínas de Transporte/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Immunoblotting , Fosforilação , Estabilidade Proteica , Inibidores da Síntese de Proteínas/farmacologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismoRESUMO
Aberrant regulation of metabolic kinases by altered redox homeostasis substantially contributes to aging and various diseases, such as diabetes. We found that the catalytic activity of a conserved family of fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases, is regulated by redox-sensitive cysteine residues in the kinase domain. The crystal structure of the FN3K homolog from Arabidopsis thaliana revealed that it forms an unexpected strand-exchange dimer in which the ATP-binding P-loop and adjoining ß strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained interchain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirmed that the strained disulfides function as redox "switches" to reversibly regulate the activity and dimerization of FN3K. Human FN3K, which contains an equivalent P-loop Cys, was also redox sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, were not. Furthermore, CRISPR-mediated knockout of FN3K in human liver cancer cells altered the abundance of redox metabolites, including an increase in glutathione. We propose that redox regulation evolved in FN3K homologs in response to changing cellular redox conditions. Our findings provide insights into the origin and evolution of redox regulation in the protein kinase superfamily and may open new avenues for targeting human FN3K in diabetic complications.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Humanos , Oxirredução , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Conformação Proteica em Folha beta , Domínios ProteicosRESUMO
The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein. Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Citosol/metabolismo , Glicosilação , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Transporte ProteicoRESUMO
Basic winged bean agglutinin binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A-reactive and B-reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A-trisaccharides and B-trisaccharides and with a variant of the A-trisaccharide have been modeled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared with the interactions of the B-trisaccharide with the lectin, the third sugar residue of the A-reactive trisacharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity.
Assuntos
Albuminas/química , Antígenos de Grupos Sanguíneos , Carboidratos/química , Proteínas de Plantas/química , Cristalografia , Dimerização , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Proteínas de Armazenamento de SementesRESUMO
Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the beta-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.
Assuntos
Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Dobramento de Proteína , Catálise , Dicroísmo Circular , Glicosilação , Hidrólise , Desnaturação Proteica , Estrutura Secundária de ProteínaRESUMO
The crystal structure of winged bean basic agglutinin in complex with GalNAc-alpha-O-Ser (Tn-antigen) has been elucidated at 2.35 angstroms resolution in order to characterize the mode of binding of Tn-antigen with the lectin. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the complexation of the lectin with Tn-antigen. Modeling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein.
Assuntos
Antígenos Glicosídicos Associados a Tumores/metabolismo , Cristalografia por Raios X , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Termodinâmica , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Simulação por Computador , Dimerização , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lectinas/química , Lectinas/metabolismo , Modelos Moleculares , Sensibilidade e Especificidade , Análise Espectral Raman , Especificidade por Substrato , Água/químicaRESUMO
Human African trypanosomiasis is caused by the eukaryotic microbe Trypanosoma brucei. To discover new drugs against the disease, one may use drugs in the clinic for other indications whose chemical scaffolds can be optimized via a medicinal chemistry campaign to achieve greater potency against the trypanosome. Towards this goal, we tested inhibitors of human EGFR and/or VEGFR as possible anti-trypanosome compounds. The 4-anilinoquinazolines canertinib and lapatinib, and the pyrrolopyrimidine AEE788 killed bloodstream T. brucei in vitro with GI(50) in the low micromolar range. Curiously, the genome of T. brucei does not encode EGFR or VEGFR, indicating that the drugs recognize alternate proteins. To discover these novel targets, a trypanosome lysate was adsorbed to an ATP-sepharose matrix and washed with a high salt solution followed by nicotinamide adenine dinucleotide (NAD(+)). Proteins that remained bound to the column were eluted with drugs, and identified by mass spectrometry/bioinformatics. Lapatinib bound to Tb927.4.5180 (termed T. brucei lapatinib-binding protein kinase-1 (TbLBPK1)) while AEE788 bound Tb927.5.800 (TbLBPK2). When the NAD(+) wash was omitted from the protocol, AEE788, canertinib and lapatinib eluted TbLBPK1, TbLBPK2, and Tb927.3.1570 (TbLBPK3). In addition, both canertinib and lapatinib eluted Tb10.60.3140 (TbLBPK4), whereas only canertinib desorbed Tb10.61.1880 (TbCBPK1). Lapatinib binds to a unique conformation of protein kinases. To gain insight into the structural basis for lapatinib interaction with TbLBPKs, we constructed three-dimensional models of lapatinibâ¢TbLBPK complexes, which confirmed that TbLBPKs can adopt lapatinib-compatible conformations. Further, lapatinib, AEE788, and canertinib were docked to TbLBPKs with favorable scores. Our studies (a) present novel targets of kinase-directed drugs in the trypanosome, and (b) offer the 4-anilinoquinazoline and pyrrolopyrimidines as scaffolds worthy of medicinal chemistry and structural biology campaigns to develop them into anti-trypanosome drugs.
Assuntos
Terapia de Alvo Molecular , Proteínas Quinases/metabolismo , Quinazolinas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cromatografia de Afinidade , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Células HeLa , Humanos , Lapatinib , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Morfolinas/química , Morfolinas/farmacologia , NAD/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Purinas/química , Purinas/farmacologia , Quinazolinas/química , Homologia Estrutural de Proteína , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Crystal structures of the complexes of basic winged-bean agglutinin with the disaccharides Galalpha1-4Gal (galabiose), Galalpha1-6Glc (mellibiose) and Galalpha1-4Galbeta-Et have been determined and the complex with Galalpha1-2Gal has been modelled. The interactions of the nonreducing Gal with the lectin at the primary site are the same as those in the known complexes with disaccharides having the alpha1-->3 linkage. The second residue in Galalpha1-4Gal and Galalpha1-6Glc forms a water bridge to the lectin, while the ethyl group in Galalpha1-4Galbeta-Et makes nonpolar interactions. In complexes involving disaccharides with alpha1-3 linkages, which form part of the A and B blood-group substances, the second sugar residue forms a direct hydrogen bond to the variable loop in the binding site of the lectin. This in part explains the specificity of the lectin for the blood-group substances and also the higher affinity of alpha1-->3-linked disaccharides for the lectin compared with disaccharides involving other linkages. Including those reported here, 14 crystal structures involving the lectin, accounting for 54 crystallographically independent subunits, are available. A comparative study of these structures shows that the region involving the curved beta-sheet which nestles the metal ions is relatively rigid. The carbohydrate-binding region is perched on this region. The flat beta-sheet, which is involved in oligomerization and exhibits considerable variability in legume lectins, is relatively flexible. Indeed, the structures of basic winged-bean lectin have been of critical importance in establishing legume lectins as a family of proteins in which small alterations in essentially the same tertiary structure lead to large variations in quaternary association. They have also provided a structural explanation of the blood-group specificity of the lectin.
Assuntos
Dissacarídeos/química , Lectinas de Plantas/química , Sítios de Ligação , Cristalografia por Raios X , Glicoproteínas/química , Melibiose/química , Modelos Moleculares , Conformação ProteicaRESUMO
The crystal structure of the complexes of basic winged-bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-alpha-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of the complexes. In addition, an N-H...O hydrogen bond involving the hydroxyl group at C2 exists in the galactose and 2-methoxygalactose complexes. An additional hydrophobic interaction involving the methyl group in the latter leads to the higher affinity of the methyl derivative. In the lectin-N-acetylgalactosamine complex the N-H...O hydrogen bond is lost, but a compensatory hydrogen bond is formed involving the O atom of the acetamido group. In addition, the CH(3) moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group alpha-linked to the galactose takes part in additional hydrophobic interactions. Therefore, methyl-alpha-N-acetylgalactosamine has a higher affinity than N-acetylgalactosamine for the lectin. The structures of basic winged-bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood-group specificity of basic winged-bean lectin.