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1.
bioRxiv ; 2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38187560

RESUMO

Inhibition of Bruton's tyrosine kinase (BTK) has proven to be highly effective in the treatment of B-cell malignancies such as chronic lymphocytic leukemia (CLL), autoimmune disorders and multiple sclerosis. Since the approval of the first BTK inhibitor (BTKi), Ibrutinib, several other inhibitors including Acalabrutinib, Zanubrutinib, Tirabrutinib and Pirtobrutinib have been clinically approved. All are covalent active site inhibitors, with the exception of the reversible active site inhibitor Pirtobrutinib. The large number of available inhibitors for the BTK target creates challenges in choosing the most appropriate BTKi for treatment. Side-by-side comparisons in CLL have shown that different inhibitors may differ in their treatment efficacy. Moreover, the nature of the resistance mutations that arise in patients appears to depend on the specific BTKi administered. We have previously shown that Ibrutinib binding to the kinase active site causes unanticipated long-range effects on the global conformation of BTK (Joseph, R.E., et al., 2020, https://doi.org/10.7554/eLife.60470 ). Here we show that binding of each of the five approved BTKi to the kinase active site brings about distinct allosteric changes that alter the conformational equilibrium of full-length BTK. Additionally, we provide an explanation for the resistance mutation bias observed in CLL patients treated with different BTKi and characterize the mechanism of action of two common resistance mutations: BTK T474I and L528W.

2.
J Mol Biol ; 434(5): 167422, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34954235

RESUMO

Mutations in PLCγ, a substrate of the tyrosine kinase BTK, are often found in patients who develop resistance to the BTK inhibitor Ibrutinib. However, the mechanisms by which these PLCγ mutations cause Ibrutinib resistance are unclear. Under normal signaling conditions, BTK mediated phosphorylation of Y783 within the PLCγ cSH2-linker promotes the intramolecular association of this site with the adjacent cSH2 domain resulting in active PLCγ. Thus, the cSH2-linker region in the center of the regulatory gamma specific array (γSA) of PLCγ is a key feature controlling PLCγ activity. Even in the unphosphorylated state this linker exists in a conformational equilibrium between free and bound to the cSH2 domain. The position of this equilibrium is optimized within the properly regulated PLCγ enzyme but may be altered in the context of mutations. We therefore assessed the conformational status of four resistance associated mutations within the PLCγ γSA and find that they each alter the conformational equilibrium of the γSA leading to a shift toward active PLCγ. Interestingly, two distinct modes of mutation induced activation are revealed by this panel of Ibrutinib resistance mutations. These findings, along with the recently determined structure of fully autoinhibited PLCγ, provide new insight into the nature of the conformational change that occurs within the γSA regulatory region to affect PLCγ activation. Improving our mechanistic understanding of how B cell signaling escapes Ibrutinib treatment via mutations in PLCγ will aid in the development of strategies to counter drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fosfolipase C gama , Piperidinas , Inibidores de Proteínas Quinases , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/genética , Tirosina Quinase da Agamaglobulinemia/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Fosfolipase C gama/química , Fosfolipase C gama/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia
3.
Elife ; 92020 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-33226337

RESUMO

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.


Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton's tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme (which allows it to modify other proteins by adding a 'tag' known as a phosphate group). The other regions of BTK, known as regulatory modules, control this activity. In BTK's inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain. Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein. Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better. Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Domínio Catalítico , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Adenina/farmacologia , Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/genética , COVID-19/metabolismo , COVID-19/prevenção & controle , COVID-19/virologia , Dasatinibe/química , Dasatinibe/metabolismo , Dasatinibe/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/prevenção & controle , Modelos Moleculares , Estrutura Molecular , Mutação , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , SARS-CoV-2/fisiologia , Domínios de Homologia de src/genética
4.
Proc Natl Acad Sci U S A ; 116(43): 21539-21544, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31591208

RESUMO

The pleckstrin homology (PH) domain is well known for its phospholipid targeting function. The PH-TEC homology (PHTH) domain within the TEC family of tyrosine kinases is also a crucial component of the autoinhibitory apparatus. The autoinhibitory surface on the PHTH domain has been previously defined, and biochemical investigations have shown that PHTH-mediated inhibition is mutually exclusive with phosphatidylinositol binding. Here we use hydrogen/deuterium exchange mass spectrometry, nuclear magnetic resonance (NMR), and evolutionary sequence comparisons to map where and how the PHTH domain affects the Bruton's tyrosine kinase (BTK) domain. The data map a PHTH-binding site on the activation loop face of the kinase C lobe, suggesting that the PHTH domain masks the activation loop and the substrate-docking site. Moreover, localized NMR spectral changes are observed for non-surface-exposed residues in the active site and on the distal side of the kinase domain. These data suggest that the association of PHTH induces allosteric conformational shifts in regions of the kinase domain that are critical for catalysis. Through statistical comparisons of diverse tyrosine kinase sequences, we identify residues unique to BTK that coincide with the experimentally determined PHTH-binding surface on the kinase domain. Our data provide a more complete picture of the autoinhibitory conformation adopted by full-length TEC kinases, creating opportunities to target the regulatory domains to control the function of these kinases in a biological setting.


Assuntos
Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Tirosina Quinase da Agamaglobulinemia/genética , Regulação Alostérica , Sítios de Ligação , Humanos , Metabolismo dos Lipídeos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Domínios de Homologia à Plecstrina , Domínios Proteicos , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo
5.
J Biol Chem ; 294(42): 15480-15494, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31484725

RESUMO

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Células HEK293 , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/genética , Proto-Oncogene Mas , Domínios de Homologia de src
6.
Structure ; 25(10): 1481-1494.e4, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28867612

RESUMO

Capturing the functionally relevant forms of dynamic, multidomain proteins is extremely challenging. Bruton's tyrosine kinase (BTK), a kinase essential for B and mast cell function, has stubbornly resisted crystallization in its full-length form. Here, nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry show that BTK adopts a closed conformation in dynamic equilibrium with open, active conformations. BTK lacks the phosphotyrosine regulatory tail of the SRC kinases, yet nevertheless achieves a phosphotyrosine-independent C-terminal latch. The unique proline-rich region is an internal "on" switch pushing the autoinhibited kinase toward its active state. Newly identified autoinhibitory contacts in the BTK pleckstrin homology domain are sensitive to phospholipid binding, which induces large-scale allosteric changes. The multiplicity of these regulatory contacts suggests a clear mechanism for gradual or "analog" kinase activation as opposed to a binary "on/off" switch. The findings illustrate how previously modeled information for recalcitrant full-length proteins can be expanded and validated with a convergent multidisciplinary experimental approach.


Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Domínio Catalítico , Medição da Troca de Deutério , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Fosfotirosina/metabolismo , Conformação Proteica , Domínios Proteicos
7.
Biochemistry ; 56(23): 2938-2949, 2017 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-28516764

RESUMO

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.


Assuntos
Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Quinases/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Estabilidade Enzimática , Bicamadas Lipídicas/química , Camundongos , Mutagênese Sítio-Dirigida , Mutação , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosforilação , Domínios de Homologia à Plecstrina , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Transporte Proteico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
8.
J Inorg Biochem ; 150: 139-47, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26141377

RESUMO

Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction.


Assuntos
Proteínas de Bactérias/química , Hemina/análogos & derivados , Hemoglobinas/química , Hidroxilamina/metabolismo , Proteínas de Plantas/química , Amônia/química , Animais , Deutério , Hemina/química , Cavalos , Cinética , Modelos Químicos , Mioglobina/química , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Oryza , Oxirredução , Espectrofotometria/métodos , Synechocystis
9.
J Biol Chem ; 290(20): 12868-78, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25839233

RESUMO

Phox/Bem1p (PB1) domains are universal structural modules that use surfaces of different charge for protein-protein association. In plants, PB1-mediated interactions of auxin response factors (ARF) and auxin/indole 3-acetic acid inducible proteins regulate transcriptional events modulated by the phytohormone auxin. Here we investigate the thermodynamic and structural basis for Arabidopsis thaliana ARF7 PB1 domain self-interaction. Isothermal titration calorimetry and NMR experiments indicate that key residues on both the basic and acidic faces of the PB1 domain contribute to and organize coordinately to stabilize protein-protein interactions. Calorimetric analysis of ARF7PB1 site-directed mutants defines a two-pronged electrostatic interaction. The canonical PB1 interaction between a lysine and a cluster of acidic residues provides one prong with an arginine and a second cluster of acidic residues defining the other prong. Evolutionary conservation of this core recognition feature and other co-varying interface sequences allows for versatile PB1-mediated interactions in auxin signaling.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Ácidos Indolacéticos , Fatores de Transcrição/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
ACS Chem Biol ; 10(1): 262-8, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25248068

RESUMO

Kinases control many aspects of cellular signaling and are therefore therapeutic targets for numerous disease states. Monitoring the conformational changes that drive activation and inactivation of the catalytic kinase core is a challenging experimental problem due to the dynamic nature of these enzymes. We apply [(13)C] reductive methylation to chemically introduce NMR-active nuclei into unlabeled protein kinases. The results demonstrate that solution NMR spectroscopy can be used to monitor specific changes in the chemical environment of structurally important lysines in a [(13)C]-methylated kinase as it shifts from the inactive to active state. This approach provides a solution based method to complement X-ray crystallographic data and can be applied to nearly any kinase, regardless of size or method of production.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Domínio Catalítico , Ativação Enzimática , Metilação , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/genética
11.
J Mol Biol ; 426(21): 3656-69, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25193673

RESUMO

Despite high level of homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamics simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest that this isoleucine side chain in the Tec family kinases acts as a "wedge" that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain.


Assuntos
Isoleucina/química , Proteínas Tirosina Quinases/química , Tirosina Quinase da Agamaglobulinemia , Catálise , Domínio Catalítico , Escherichia coli/enzimologia , Leucina/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Simulação de Dinâmica Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Domínios de Homologia de src/genética
12.
RNA ; 20(6): 815-24, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24757168

RESUMO

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.


Assuntos
Aptâmeros de Nucleotídeos/química , Framicetina/química , RNA/química , 2-Aminopurina/química , Aminoglicosídeos/química , Sítios de Ligação , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Conformação de Ácido Nucleico , Concentração Osmolar , Especificidade por Substrato
13.
Sci Signal ; 6(290): ra76, 2013 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-23982207

RESUMO

Itk (interleukin-2-inducible T cell kinase) and Btk (Bruton's tyrosine kinase) are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of 6 of the 619 amino acid residues of Itk with the corresponding residues of Btk (and vice versa) was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties, depending on the cellular context. We suggest that the weaker catalytic activities of T cell-specific kinases serve to regulate cellular activation and prevent aberrant immune responses.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Western Blotting , Medição da Troca de Deutério , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Homologia de Sequência de Aminoácidos , Células Sf9 , Linfócitos T/metabolismo , Tirosina/genética , Tirosina/metabolismo
14.
J Mol Biol ; 425(4): 683-96, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23219468

RESUMO

Itk (interleukin-2 inducible T cell kinase) is a non-receptor protein tyrosine kinase expressed primarily in T cells. Itk catalyzes phosphorylation on tyrosine residues within a number of its natural substrates, including the well-characterized Y783 of PLCγ1. However, the molecular mechanisms Itk exploits to recognize its substrates are not completely understood. We have previously identified a specific docking interaction between the kinase domain of Itk and the C-terminal Src homology 2 (SH2C) domain of PLCγ1 that promotes substrate specificity for this enzyme/substrate pair. In the current study, we identify and map the interaction surface on the Itk kinase domain as an acidic patch centered on the G helix. Mutation of the residues on and adjacent to the G helix within the Itk kinase domain impairs the catalytic efficacy of PLCγ1 substrate phosphorylation by specifically altering the protein-protein interaction interface and not the inherent catalytic activity of Itk. NMR titration experiments using a Btk (Bruton's tyrosine kinase) kinase domain as a surrogate for the Itk kinase domain provide further support for an Itk/PLCγ1 SH2C interaction surrounding the G helix of the kinase domain. The work presented here provides structural insight into how the Itk kinase uses the G helix to single out Y783 of PLCγ1 for specific phosphorylation. Comparing these results to other well-characterized kinase/substrate systems suggests that the G helix is a general structural feature used by kinases for substrate recognition during signaling.


Assuntos
Fosfolipase C gama/química , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Western Blotting , Cinética , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeos/química , Peptídeos/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Especificidade por Substrato , Tirosina/química , Tirosina/genética , Tirosina/metabolismo , Domínios de Homologia de src/genética
15.
Protein Sci ; 21(9): 1288-97, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22761113

RESUMO

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.


Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade
16.
Artigo em Inglês | MEDLINE | ID: mdl-22297986

RESUMO

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the ß-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively.


Assuntos
Proteínas Tirosina Quinases/química , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Proteínas Tirosina Quinases/genética
17.
Biochemistry ; 50(50): 10829-35, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22080728

RESUMO

Plants often face hypoxic stress as a result of flooding and waterlogged soils. During these periods, they must continue ATP production and nitrogen metabolism if they are to survive. The normal pathway of reductive nitrogen assimilation in non-legumes, nitrate, and nitrite reductase can be inhibited during low oxygen conditions that are associated with the buildup of toxic metabolites such as nitrite and nitric oxide, so the plant must also have a means of detoxifying these molecules. Compared to animal hemoglobins, plant and cyanobacterial hemoglobins are adept at reducing nitrite to nitric oxide under anaerobic conditions. Here we test their abilities to reduce hydroxylamine, a proposed intermediate of nitrite reductase, under anaerobic conditions. We find that class 1 rice nonsymbiotic hemoglobin (rice nsHb1) and the hemoglobin from the cyanobacterium Synechocystis (SynHb) catalyze the reduction of hydroxylamine to ammonium at rates 100-2500 times faster than animal hemoglobins including myoglobin, neuroglobin, cytoglobin, and blood cell hemoglobin. These results support the hypothesis that plant and cyanobacterial hemoglobins contribute to anaerobic nitrogen metabolism in support of anaerobic respiration and survival during hypoxia.


Assuntos
Proteínas de Bactérias/metabolismo , Globinas/metabolismo , Hemoglobinas/metabolismo , Hidroxilamina/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Compostos de Amônio Quaternário/metabolismo , Synechocystis/metabolismo , Hemoglobinas Truncadas/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citoglobina , Globinas/química , Globinas/genética , Hemoglobinas/química , Hemoglobinas/genética , Cavalos , Humanos , Cinética , Mioglobina/química , Mioglobina/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neuroglobina , Ressonância Magnética Nuclear Biomolecular , Oryza/enzimologia , Oxirredução , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Synechocystis/enzimologia , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genética
18.
Biochem J ; 435(3): 589-95, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21323642

RESUMO

The evolution of natural product biosynthetic pathways can be envisioned to occur via a number of mechanisms. In the present study we provide evidence that latent plasticity plays a role in such metabolic evolution. In particular, rice (Oryza sativa) produces both ent- and syn-CPP (copalyl diphosphate), which are substrates for downstream diterpene synthases. In the present paper we report that several members of this enzymatic family exhibit dual reactivity with some pairing of ent-, syn- or normal CPP stereochemistry. Evident plasticity was observed, as a previously reported ent-sandaracopimaradiene synthase also converts syn-CPP into syn-labda-8(17),12E,14-triene, which can be found in planta. Notably, normal CPP is not naturally found in rice. Thus the presence of diterpene synthases that react with this non-native metabolite reveals latent enzymatic/metabolic plasticity, providing biochemical capacity for utilization of such a novel substrate (i.e. normal CPP) which may arise during evolution, the implications of which are discussed.


Assuntos
Alquil e Aril Transferases/metabolismo , Evolução Biológica , Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/genética , Regulação Enzimológica da Expressão Gênica , Estrutura Molecular , Organofosfatos/química , Organofosfatos/metabolismo , Oryza/genética , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Estereoisomerismo , Especificidade por Substrato
19.
J Phys Chem A ; 115(16): 3630-41, 2011 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-20446696

RESUMO

We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.


Assuntos
Apoproteínas/química , Cumarínicos/química , Fluorescência , Simulação de Dinâmica Molecular , Mioglobina/química , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Molecular
20.
J Am Chem Soc ; 132(50): 17680-3, 2010 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-21117645

RESUMO

Coordinatively saturated To(M)MgMe (1; To(M) = tris(4,4-dimethyl-2-oxazolinyl)phenylborate) is an active precatalyst for intramolecular hydroamination/cyclization at 50 °C. The empirical rate law of -d[substrate]/dt = k'(obs)[Mg](1)[substrate](1) and Michaelis-Menten-type kinetics are consistent with a mechanism involving reversible catalyst-substrate association prior to cyclization. The resting state of the catalyst, To(M)MgNHCH(2)CR(2)CH(2)CH═CH(2) [R = Ph, Me, -(CH(2))(5)-], is isolable, but isolated magnesium amidoalkene does not undergo unimolecular cyclization at 50 °C. However, addition of trace amounts of substrate allows cyclization to occur. Therefore, we propose a two-substrate, six-center transition state involving concerted C-N bond formation and N-H bond cleavage as the turnover-limiting step of the catalytic cycle.

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