RESUMEN
In line with their high accessibility, disordered proteins are exquisite targets of kinases. Eukaryotic organisms use the so-called intrinsically disordered proteins (IDPs) or intrinsically disordered regions of proteins (IDRs) as molecular switches carrying intracellular information tuned by reversible phosphorylation schemes. Solvent-exposed serines and threonines are abundant in IDPs, and, consistently, kinases often modify disordered regions of proteins at multiple sites. In this context, nuclear magnetic resonance (NMR) spectroscopy provides quantitative, residue-specific information that permits mapping of phosphosites and monitoring of their individual kinetics. Hence, NMR monitoring emerges as an in vitro approach, complementary to mass-spectrometry or immuno-blotting, to characterize IDP phosphorylation comprehensively. Here, we describe in detail generic protocols for carrying out NMR monitoring of IDP phosphorylation, and we provide a number of practical insights that improve handiness and reproducibility of this method.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Procesamiento Proteico-Postraduccional , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Resonancia Magnética Nuclear Biomolecular/instrumentación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Fosfoserina/química , Fosfotreonina/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Quinasa Tipo Polo 1RESUMEN
ErbB4 receptor tyrosine kinase has four different isoforms that are classified based on variants in the extracellular juxtamembrane domain (JM-a and JM-b) and the C-terminal region (CYT-1 and CYT-2). Here, we used the JM-b/CYT-1 isoform to investigate the roles of serine/threonine phosphorylation in MEK-ERK-dependent feedback inhibition. TPA as an activator of the ERK pathway markedly induced ErbB4 phosphorylation at Thr-674, the conserved common feedback site in the intracellular JM domain, which resulted in the downregulation of tyrosine autophosphorylation. We also identified Ser-1026 as an ErbB4-specific ERK target site in the CYT-1 region. Moreover, double mutations (Thr-674/Ser-1026 to Ala) significantly upregulated ErbB4 activation, indicating that Thr-674 and Ser-1026 are cooperatively involved in negative feedback regulation. Given the fact that ErbB4 mutation is one of the most common genetic alterations in melanoma cells, we demonstrated that a typical oncogenic ErbB4 mutant was resistant to the negative feedback regulation to maintain a highly active status of tyrosine kinase activity. Together, these findings indicate that feedback mechanisms are key switches determining oncogenic potentials of ErbB receptor kinases.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Retroalimentación Fisiológica , Receptor ErbB-4/química , Receptor ErbB-4/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Melanoma/genética , Mutación , Fosforilación , Fosfoserina/química , Fosfoserina/metabolismo , Fosfotreonina/química , Fosfotreonina/metabolismo , Receptor ErbB-4/genéticaRESUMEN
Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks (Plk1-5), Plk1 is recognized as an anticancer drug target. Plk1 contains multiple structural components that are important for its proper biological function. These include an N-terminal catalytic domain and a C-terminal non-catalytic polo-box domain (PBD). The PBD binds to phosphothreonine (pT) and phosphoserine-containing sequences. Blocking PBD-dependent interactions offers a potential means of down-regulating Plk1 function that is distinct from targeting its ATP-binding site. Previously, we demonstrated by tethering alkylphenyl chains from the N(π)-position of the His residue in the 5-mer PLHSpT, that we were able to access a hydrophobic "cryptic" binding pocket on the surface of the PBD, and in so doing enhance binding affinities by approximately 1000-fold. More recently, we optimized these PBD-ligand interactions using an oxime ligation-based strategy. Herein, using azide-alkyne cycloaddition reactions, we explore new triazole-containing PBD-binding antagonists. Some of these ligands retain the high PBD-binding affinity of the parent peptide, while showing desirable enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Péptidos , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Péptidos/síntesis química , Péptidos/farmacología , Fosfoserina/química , Fosfotreonina/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Triazoles , Quinasa Tipo Polo 1RESUMEN
We herein report two examples of one-pot, simultaneous reactions, mediated by multiple, orthogonal catalysts with the same catalytic motif. First, BINOL-derived chiral phosphoric acids (CPA) and phosphothreonine (pThr)-embedded peptides were found to be matched for two different steps in double reductions of bisquinolines. Next, two π-methylhistidine (Pmh)-containing peptides catalyzed enantio- and chemoselective acylations and phosphorylations of multiple substrates in one pot. The selectivity exhibited by common reactive moieties is adjusted solely by the appended chiral scaffold through outer-sphere interactions.
Asunto(s)
Péptidos/química , Ácidos Fosfóricos/química , Fosfotreonina/química , Catálisis , Estructura Molecular , NaftolesRESUMEN
Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phosphothreonine 231 (pThr-231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved >20-fold over that of the WT antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity toward the nonphosphorylated target site but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phosphotau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies and highlight the importance of assessing the specificity in the antibody engineering process.
Asunto(s)
Evolución Molecular Dirigida/métodos , Fosfotreonina/química , Procesamiento Proteico-Postraduccional , Anticuerpos de Cadena Única/química , Proteínas tau/química , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Sitios de Unión , Técnicas de Visualización de Superficie Celular , Reacciones Cruzadas , Expresión Génica , Humanos , Modelos Moleculares , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Ingeniería de Proteínas , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Anticuerpos de Cadena Única/biosíntesis , Proteínas tau/genética , Proteínas tau/inmunología , Proteínas tau/metabolismoRESUMEN
Transcription factor c-Myc is an oncoprotein that is regulated at the post-translational level through phosphorylation of two conserved residues, Serine 62 (Ser62) and Threonine 58 (Thr58). A highly specific tool capable of recognizing Myc via pThr58 is needed to monitor activation and localization. Through phage display, we have isolated 10 engineered Forkhead-associated (FHA) domains that selectively bind to a phosphothreonine (pThr)-containing peptide (53-FELLPpTPPLSPS-64) segment of human c-Myc. One domain variant was observed to bind to the Myc-pThr58 peptide with a KD value of 800â¯nM and had >1000-fold discrimination between the phosphorylated and non-phosphorylated peptide. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its ß4-ß5 and ß10-ß11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. The Myc-pTBD's specificity for its cognate ligand was demonstrated to be on a par with 3 commercial polyclonal antibodies, suggesting that this recombinant reagent is a viable alternative to antibodies for monitoring Myc regulation.
Asunto(s)
Fosfopéptidos/química , Fosfotreonina/química , Proteínas Proto-Oncogénicas c-myc/química , Humanos , Modelos Moleculares , Fosfopéptidos/metabolismo , Fosfotreonina/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Among all the post-translational modifications (PTMs) of proteins, Phosphorylation is known to be the most important and highly occurring PTM in eukaryotes and prokaryotes. It has an important regulatory mechanism which is required in most of the pathological and physiological processes including neural activity and cell signalling transduction. The process of threonine phosphorylation modifies the threonine by the addition of a phosphoryl group to the polar side chain, and generates phosphothreonine sites. The investigation and prediction of phosphorylation sites is important and various methods have been developed based on high throughput mass-spectrometry but such experimentations are time consuming and laborious therefore, an efficient and accurate novel method is proposed in this study for the prediction of phosphothreonine sites. The proposed method uses context-based data to calculate statistical moments. Position relative statistical moments are combined together to train neural networks. Using 10-fold cross validation, 94.97% accurate result has been obtained whereas for Jackknife testing, 96% accurate results have been obtained. The overall accuracy of the system is 94.4% to sensitivity value 94% and specificity 94.6%. These results suggest that the proposed method may play an essential role to the other existing methods for phosphothreonine sites prediction.
Asunto(s)
Fosfoproteínas , Fosfotreonina/química , Análisis de Secuencia de Proteína/métodos , Programas Informáticos , Fosfoproteínas/química , Fosfoproteínas/genética , FosforilaciónRESUMEN
Phosphothreonine (pThr)-embedded peptide catalysts are found to mediate the reductive amination of 3-amidocyclohexanones with divergent selectivity. The choice of peptide sequence can be used to alter the diastereoselectivity to favor either the cis-product or trans-product, which are obtained in up to 93:7 er. NMR studies and DFT calculations are reported and indicate that both pathways rely on secondary interactions between substrate and catalyst to achieve selectivity. Furthermore, catalysts appear to accomplish a parallel kinetic resolution of the substrates. The facility for phosphopeptides to tune reactivity and access multiple products in reductive aminations may translate to the diversification of complex substrates, such as natural products, at numerous reactive sites.
Asunto(s)
Productos Biológicos/síntesis química , Ciclohexanonas/química , Fosfotreonina/química , Aminación , Productos Biológicos/química , Catálisis , Cinética , Estructura Molecular , Oxidación-Reducción , Teoría Cuántica , EstereoisomerismoRESUMEN
One of the most common chemistries used to label primary amines utilizes N-hydroxysuccinimide (NHS), which is also structurally incorporated in various quantitative proteomic reagents such as isobaric tags for relative and absolute quantification (iTRAQ) and tandem mass tags (TMT). In this paper we report detrimental effect of hydroxylamine, a widely used quenching reagent for excess NHS, on phosphopeptides. We found an impairment in the degree of phosphopeptide identification when hydroxylamine-quenched TMT-labeled samples were vacuum-dried and desalted compared to the nondried (just diluted) and desalted ones prior to phosphoenrichment. We have also demonstrated that vacuum-drying in the presence of hydroxylamine promotes ß-elimination of phosphate groups from phosphoserine and phosphothreonine while having a minimalistic effect on phosphotyrosine. Additionally, we herein report that this negative impact of hydroxylamine could be minimized by direct desalting after appropriate dilution of quenched samples. We also found a 1.6-fold increase in the number of phosphopeptide identifications after employing our optimized method. The above method was also successfully applied to human tumor tissues to quantify over 15000 phosphopeptides from 3 mg TMT 6-plex labeled-peptides.
Asunto(s)
Hidroxilamina/química , Indicadores y Reactivos/química , Fosfopéptidos/análisis , Proteómica/métodos , Succinimidas/química , Humanos , Fosfopéptidos/química , Fosfoserina/química , Fosfotreonina/químicaRESUMEN
BACKGROUND: CDK6 is considered as a highly validated anticancer drug target due to its essential role in regulating cell cycle progression at G1 restriction point. Activation of CDK6 requires the phosphorylation of Thr177 on A-loop, but the structural insights of the activation mechanism remain unclear. METHODS: Herein, all-atoms molecular dynamics (MD) simulations were used to study the effects of Thr177 phosphorylation on the dynamic structure of CDK6-Vcyclin complex. RESULTS: MD results indicated that the free energy barrier of the transition from open to closed state decreased ~47.2% after Thr177 phosphorylation. Key steps along the state transition process were obtained from a cluster analysis. Binding preference of ten different inhibitors to open or closed state were also investigated through molecular docking along with MD simulations methods. CONCLUSIONS: Our results indicated that Thr177 phosphorylation increased the flexibility around the ATP-binding pocket. The transition of the ATP-binding pocket between open and closed states should be considered for understanding the binding of CDK6 inhibitors. GENERAL SIGNIFICANCE: This work could deepen the understanding of CDKs activation mechanism, and provide useful information for the discovery of new CDKs inhibitors with high affinity and specificity.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/química , Fosfotreonina/química , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Sitios de Unión , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Activación Enzimática , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
The dual specificity phosphatase DUSP1 was the first mitogen activated protein kinase phosphatase (MKP) to be identified. It dephosphorylates conserved tyrosine and threonine residues in the activation loops of mitogen activated protein kinases ERK2, JNK1 and p38-alpha. Here, we report the crystal structure of the human DUSP1 catalytic domain at 2.49 Å resolution. Uniquely, the protein was crystallized as an MBP fusion protein in complex with a monobody that binds to MBP. Sulfate ions occupy the phosphotyrosine and putative phosphothreonine binding sites in the DUSP1 catalytic domain.
Asunto(s)
Fosfatasa 1 de Especificidad Dual/química , Fosfatasa 1 de Especificidad Dual/metabolismo , Proteínas de Unión a Maltosa/metabolismo , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Fosfotreonina/química , Fosfotirosina/química , Conformación Proteica , Especificidad por Sustrato , Sulfatos/químicaRESUMEN
The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de la Membrana/química , Proteínas Asociadas a Microtúbulos/química , Fosfoserina/química , Fosfotreonina/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Homólogo 4 de la Proteína Discs Large , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutatión Transferasa/química , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Imitación Molecular , Fosfoserina/metabolismo , Fosfotreonina/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
(2S,3R)-2-Amino-3-methyl-4-phosphonobutanoic acid (Pmab) is a phosphatase-stable analogue of phosphothreonine (pThr), which has been used in a variety of biological contexts. Among these applications are peptidomimetic ligands that bind to the polo-box domain (PBD) of polo-like kinase 1 (Plk1) with affinities approaching that of the corresponding pThr-containing peptides. However, Pmab is not widely used, because there are no direct, high-yield preparations of suitably protected reagent. We have now achieved an efficient synthesis of protected Pmab, as well as variants with different substituents at the 3R center. When incorporated into our peptidomimetic scaffold, these new Pmab analogues exhibit Plk1 PBD-binding affinities that are several-fold higher than Pmab, yet retain good selectivity for Plk1 relative to the PBDs of Plk2 and Plk3. These findings will significantly impact the future development of PBD-binding inhibitors, as well as ligands directed against a broad spectrum of pThr-dependent processes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ácidos Fosfoaminos/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Ácidos Fosfoaminos/metabolismo , Fosfotreonina/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Relación Estructura-Actividad , Quinasa Tipo Polo 1RESUMEN
Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such post-translational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases, including human Myc, calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2). The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). The FHA domains binding Myc, CaMKII, and ERK1/2 were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains.
Asunto(s)
Péptidos/química , Fosfotreonina/química , Marcadores de Afinidad , Secuencia de Aminoácidos , Anticuerpos , Biotecnología , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Modelos Moleculares , Biblioteca de Péptidos , Péptidos/genética , Péptidos/inmunología , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra ß-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos/metabolismo , Fosfotreonina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Fosfotreonina/química , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
Phosphothreonine (pThr) was found to constitute a new class of chiral phosphoric acid (CPA) catalyst upon insertion into peptides. To demonstrate the potential of these phosphopeptides as asymmetric catalysts, enantioselective transfer hydrogenations of a previously underexplored substrate class for CPA-catalyzed reductions were carried out. pThr-containing peptides lead to the observation of enantioselectivities of up to 94:6 e.r. with 2-substituted quinolines containing C8-amino functionality. NMR studies indicate that hydrogen-bonding interactions promote strong complexation between substrates and a rigid ß-turn catalyst.
Asunto(s)
Aminoquinolinas/química , Hidrógeno/química , Péptidos/química , Fosfotreonina/química , Dominio CatalíticoRESUMEN
Detection of protein phosphorylation at a specific residue has been achieved by using antibodies, which have usually been raised by animal immunization. However, there have been no reports of the humanization of phosphospecific non-human antibodies. Here, we report the humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at residue 231 (pT231 peptide) as a model for better understanding the phosphoepitope recognition mechanism. In the chicken antibody, the phosphate group of the pT231-peptide antigen is exclusively recognized by complementarity determining region 2 of the heavy chain variable domain (VH-CDR2). Simple grafting of six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in the complete loss of pT231-peptide binding. Using a yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, we identified 5 critical FR residues. The back mutation of these residues to the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of the humanized antibody. Importantly, the back mutation of the FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. Our humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied to other animal antibodies containing unique binding motifs on CDRs specific to posttranslationally modified epitopes.
Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Fosfotreonina/inmunología , Proteínas tau/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Afinidad de Anticuerpos , Pollos , Biblioteca de Genes , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Fosfotreonina/química , Ingeniería de Proteínas , Proteínas tau/químicaRESUMEN
Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mM). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(Ni(I))·CH3SCoM) is highly favored (Kd = 79 µM). Only then can the chemical reaction occur (kobs = 20 s(-1) at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(Ni(II))·CoB7S(-)·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.
Asunto(s)
Proteínas Arqueales/metabolismo , Mesna/metabolismo , Methanobacteriaceae/enzimología , Oxidorreductasas/metabolismo , Fosfotreonina/análogos & derivados , Proteínas Arqueales/química , Proteínas Arqueales/genética , Biocatálisis , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Mesna/química , Metano/química , Metano/metabolismo , Methanobacteriaceae/genética , Modelos Biológicos , Modelos Químicos , Níquel/química , Níquel/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Fosfotreonina/química , Fosfotreonina/metabolismo , Unión Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
The four mammalian Pellinos (Pellinos 1, 2, 3a, and 3b) are E3 ubiquitin ligases that are emerging as critical mediators for a variety of immune signaling pathways, including those activated by Toll-like receptors, the T-cell receptor, and NOD2. It is becoming increasingly clear that each Pellino has a distinct role in facilitating immune receptor signaling. However, the underlying mechanisms by which these highly homologous proteins act selectively in these signaling pathways are not clear. In this study, we investigate whether Pellino substrate recognition contributes to the divergent functions of Pellinos. Substrate recognition of each Pellino is mediated by its noncanonical forkhead-associated (FHA) domain, a well-characterized phosphothreonine-binding module. Pellino FHA domains share very high sequence identity, so a molecular basis for differences in substrate recognition is not immediately apparent. To explore Pellino substrate specificity, we first identify a high-affinity Pellino2 FHA domain-binding motif in the Pellino substrate, interleukin-1 receptor-associated kinase 1 (IRAK1). Analysis of binding of the different Pellinos to a panel of phosphothreonine-containing peptides derived from the IRAK1-binding motif reveals that each Pellino has a distinct phosphothreonine peptide binding preference. We observe a similar binding specificity in the interaction of Pellinos with a number of known Pellino substrates. These results argue that the nonredundant roles that Pellinos play in immune signaling are in part due to their divergent substrate specificities. This new insight into Pellino substrate recognition could be exploited for pharmacological advantage in treating inflammatory diseases that have been linked to the aberrant regulation of Pellinos.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosfotreonina/química , Fosfotreonina/metabolismo , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos/fisiología , Animales , Cristalografía por Rayos X , Células HEK293 , Humanos , Ratones , Unión Proteica/fisiología , Especificidad por Sustrato/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.