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1.
Proc Natl Acad Sci U S A ; 121(30): e2407159121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39012820

RESUMO

Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.


Assuntos
Simulação de Dinâmica Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Domínios de Homologia de src , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Humanos , Domínios de Homologia de src/genética , Ligação Proteica , Mutação , Fosforilação , Sítios de Ligação/genética , Fosfotirosina/metabolismo , Ligantes
2.
J Am Chem Soc ; 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39024122

RESUMO

Contemporary developments in the field of peptide macrocyclization methodology are imperative for enabling the advance of drug design in medicinal chemistry. This report discloses a Rh(III)-catalyzed macrocyclization via carboamidation, reacting acryloyl-peptide-dioxazolone precursors and arylboronic acids to form complex cyclic peptides with concomitant incorporation of noncanonical α-amino acids. The diverse and modular technology allows for expedient access to a wide variety of cyclic peptides from 4 to 15 amino acids in size and features simultaneous formation of unnatural phenylalanine and tyrosine derivatives with up to >20:1 diastereoselectivity. The reaction showcases an expansive substrate scope with 45 examples and is compatible with the majority of standard protected amino acids used in Fmoc-solid phase peptide synthesis. The methodology is applied to the synthesis of multiple peptidomimetic macrocyclic analogs, including derivatives of cyclosomatostatin and gramicidin S.

3.
J Am Chem Soc ; 146(2): 1337-1345, 2024 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-38165744

RESUMO

State-of-the-art methods in photoproximity labeling center on the targeted generation and capture of short-lived reactive intermediates to provide a snapshot of local protein environments. Diazirines are the current gold standard for high-resolution proximity labeling, generating short-lived aryl(trifluoromethyl) carbenes. Here, we present a method to access aryl(trifluoromethyl) carbenes from a stable diazo source via tissue-penetrable, deep red to near-infrared light (600-800 nm). The operative mechanism of this activation involves Dexter energy transfer from photoexcited osmium(II) photocatalysts to the diazo, thus revealing an aryl(trifluoromethyl) carbene. The labeling preferences of the diazo probe with amino acids are studied, showing high reactivity toward heteroatom-H bonds. Upon the synthesis of a biotinylated diazo probe, labeling studies are conducted on native proteins as well as proteins conjugated to the Os photocatalyst. Finally, we demonstrate that the conjugation of a protein inhibitor to the photocatalyst also enables selective protein labeling in the presence of spectator proteins and achieves specific labeling of a membrane protein on the surface of mammalian cells via a two-antibody photocatalytic system.


Assuntos
Proteínas , Luz Vermelha , Animais , Proteínas/química , Metano/química , Diazometano/química , Mamíferos
4.
bioRxiv ; 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-37502916

RESUMO

Mutations in the tyrosine phosphatase SHP2 are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting auto-inhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that, while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8-10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.

5.
Immunity ; 56(12): 2682-2698.e9, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38091950

RESUMO

T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.


Assuntos
Transdução de Sinais , Linfócitos T , Ubiquitina-Proteína Ligases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Concentração de Íons de Hidrogênio
6.
Chembiochem ; 24(10): e202200706, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-36893077

RESUMO

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that modulate essential cellular processes through protein dephosphorylation and are dysregulated in various disease states. There is demand for new compounds that target the active sites of these enzymes, for use as chemical tools to dissect their biological roles or as leads for the development of new therapeutics. In this study, we explore an array of electrophiles and fragment scaffolds to investigate the required chemical parameters for covalent inhibition of tyrosine phosphatases. Our analysis juxtaposes the intrinsic electrophilicity of these compounds with their potency against several classical PTPs, revealing chemotypes that inhibit tyrosine phosphatases while minimizing excessive, potentially non-specific reactivity. We also assess sequence divergence at key residues in PTPs to explain their differential susceptibility to covalent inhibition. We anticipate that our study will inspire new strategies to develop covalent probes and inhibitors for tyrosine phosphatases.


Assuntos
Proteínas Tirosina Fosfatases , Tirosina , Domínio Catalítico , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo
7.
Elife ; 122023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36927728

RESUMO

Tyrosine kinases and SH2 (phosphotyrosine recognition) domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue. Although the preferred recognition motifs of many kinases and SH2 domains are known, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases and SH2 domains. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences. We also screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. We extended this platform to the analysis of SH2 domains and showed that screens could predict relative binding affinities. Finally, we expanded our method to assess the impact of non-canonical and post-translationally modified amino acids on sequence recognition. This specificity profiling platform will shed new light on phosphotyrosine signaling and could readily be adapted to other protein modification/recognition domains.


Assuntos
Proteínas Tirosina Quinases , Domínios de Homologia de src , Humanos , Proteínas Tirosina Quinases/metabolismo , Fosfotirosina , Fosfopeptídeos/química , Tirosina/metabolismo , Biblioteca de Peptídeos , Fosfotransferases/metabolismo
8.
bioRxiv ; 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36711903

RESUMO

The majority of cellular proteins interact with at least one partner or assemble into molecular-complexes to exert their function. This network of protein-protein interactions (PPIs) and the composition of macromolecular machines differ between cell types and physiological conditions. Therefore, characterizing PPI networks and their dynamic changes is vital for discovering novel biological functions and underlying mechanisms of cellular processes. However, producing an in-depth, global snapshot of PPIs from a given specimen requires measuring tens to thousands of LC-MS/MS runs. Consequently, while recent works made seminal contributions by mapping PPIs at great depth, almost all focused on just 1-2 conditions, generating comprehensive but mostly static PPI networks. In this study we report the development of SEC-TMT, a method that enables identifying and measuring PPIs in a quantitative manner from only 4-8 LC-MS/MS runs per biological sample. This was accomplished by incorporating tandem mass tag (TMT) multiplexing with a size exclusion chromatography mass spectrometry (SEC-MS) work-flow. SEC-TMT reduces measurement time by an order of magnitude while maintaining resolution and coverage of thousands of cellular interactions, equivalent to the gold standard in the field. We show that SEC-TMT provides benefits for conducting differential analyses to measure changes in the PPI network between conditions. This development makes it feasible to study dynamic systems at scale and holds the potential to drive more rapid discoveries of PPI impact on cellular processes.

9.
J Am Chem Soc ; 145(2): 1129-1135, 2023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36576945

RESUMO

Herein we report a modular peptide ligation methodology that couples dioxazolones, arylboronic acids, and acrylamides to construct amide bonds in a diastereoselective manner under mild conditions, facilitated by Rh(III) catalysis. By converting the C-terminus of one peptide into a dioxazolone and the N-terminus of a second peptide into an acrylamide, the two pieces can be bridged by an arylboronic acid to construct unnatural phenylalanine, tyrosine, and tryptophan residues at the junction point with diastereoselectivity for their corresponding d-stereocenters. The reaction exhibits excellent functional group tolerance with a large substrate scope and is compatible with a wide array of protected amino acid residues that are utilized in Fmoc solid phase peptide synthesis. The methodology is applied to the synthesis of six diastereomeric proteasome inhibitor analogs, as well as the ligation of two 10-mer oligopeptides to construct a 21-mer polypeptide with an unnatural phenylalanine residue at the center.


Assuntos
Aminoácidos , Peptídeos , Peptídeos/química , Aminoácidos/química , Fenilalanina , Catálise
10.
Protein Sci ; 31(10): e4411, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36173161

RESUMO

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.


Assuntos
Escherichia coli , Peptídeos e Proteínas de Sinalização Intracelular , Antibacterianos , Precursores Enzimáticos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Peptídeos/química , Fosforilação , Quinase Syk/genética , Quinase Syk/metabolismo , Tirosina/genética
11.
Nat Biotechnol ; 40(10): 1520-1527, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35606422

RESUMO

Protein-ligand interactions are increasingly profiled at high throughput using affinity selection and massively parallel sequencing. However, these assays do not provide the biophysical parameters that most rigorously quantify molecular interactions. Here we describe a flexible machine learning method, called ProBound, that accurately defines sequence recognition in terms of equilibrium binding constants or kinetic rates. This is achieved using a multi-layered maximum-likelihood framework that models both the molecular interactions and the data generation process. We show that ProBound quantifies transcription factor (TF) behavior with models that predict binding affinity over a range exceeding that of previous resources; captures the impact of DNA modifications and conformational flexibility of multi-TF complexes; and infers specificity directly from in vivo data such as ChIP-seq without peak calling. When coupled with an assay called KD-seq, it determines the absolute affinity of protein-ligand interactions. We also apply ProBound to profile the kinetics of kinase-substrate interactions. ProBound opens new avenues for decoding biological networks and rationally engineering protein-ligand interactions.


Assuntos
Aprendizado de Máquina , Fatores de Transcrição , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA/genética , Ligantes , Ligação Proteica , Fatores de Transcrição/metabolismo
12.
Elife ; 112022 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-35272765

RESUMO

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.


Assuntos
Proteínas Ativadoras de GTPase , Neoplasias , Animais , Mamíferos , Mutagênese , Mutação , Nucleotídeos , Proteínas Ativadoras de ras GTPase
13.
Protein Sci ; 30(12): 2373-2384, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34601763

RESUMO

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.


Assuntos
Simulação de Dinâmica Molecular , Quinase Syk/química , Proteína-Tirosina Quinase ZAP-70/química , Imunidade Adaptativa , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sítios de Ligação , Evolução Biológica , Clonagem Molecular , Cristalografia por Raios X , Medição da Troca de Deutério , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/imunologia
14.
Methods Mol Biol ; 2133: 31-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32144662

RESUMO

In recent years, split inteins have seen widespread use as molecular platforms for the design of a variety of peptide and protein chemistry technologies, most notably protein ligation. The development of these approaches is dependent on the identification and/or design of split inteins with robust activity, stability, and solubility. Here, we describe two approaches to characterize and compare the activities of newly identified or engineered split inteins. The first assay employs an E. coli-based selection system to rapidly screen the activities of many inteins and can be repurposed for directed evolution. The second assay utilizes reverse-phase high-performance liquid chromatography (RP-HPLC) to provide insights into individual chemical steps in the protein splicing reaction, information that can guide further engineering efforts. These techniques provide useful alternatives to common assays that utilize SDS-PAGE to analyze splicing reaction progress.


Assuntos
Clonagem Molecular/métodos , Inteínas , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas , Expressão Gênica , Inteínas/genética , Resistência a Canamicina , Processamento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas de Síntese em Fase Sólida/métodos , Trans-Splicing
15.
Nat Immunol ; 20(11): 1481-1493, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31611699

RESUMO

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Feminino , Ligantes , Masculino , Proteínas de Membrana/imunologia , Camundongos , Fosfolipase C gama/metabolismo , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/metabolismo , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo
16.
Elife ; 82019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31407665

RESUMO

Predicting ancestral sequences of protein kinases reveals the molecular details that underlie different modes of activation.


Assuntos
Proteínas Quinases
17.
Protein Sci ; 28(6): 1071-1082, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30942928

RESUMO

The multi-subunit Ca2+ /calmodulin-dependent protein kinase II (CaMKII) holoenzyme plays a critical role in animal learning and memory. The kinase domain of CaMKII is connected by a flexible linker to a C-terminal hub domain that assembles into a 12- or 14-subunit scaffold that displays the kinase domains around it. Studies on CaMKII suggest that the stoichiometry and dynamic assembly/disassembly of hub oligomers may be important for CaMKII regulation. Although CaMKII is a metazoan protein, genes encoding predicted CaMKII-like hub domains, without associated kinase domains, are found in the genomes of some green plants and bacteria. We show that the hub domains encoded by three related green algae, Chlamydomonas reinhardtii, Volvox carteri f. nagarensis, and Gonium pectoral, assemble into 16-, 18-, and 20-subunit oligomers, as assayed by native protein mass spectrometry. These are the largest known CaMKII hub domain assemblies. A crystal structure of the hub domain from C. reinhardtii reveals an 18-subunit organization. We identified four intra-subunit hydrogen bonds in the core of the fold that are present in the Chlamydomonas hub domain, but not in metazoan hubs. When six point mutations designed to recapitulate these hydrogen bonds were introduced into the human CaMKII-α hub domain, the mutant protein formed assemblies with 14 and 16 subunits, instead of the normal 12- and 14-subunit assemblies. Our results show that the stoichiometric balance of CaMKII hub assemblies can be shifted readily by small changes in sequence.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Sequência de Aminoácidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/isolamento & purificação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Alinhamento de Sequência
18.
Nat Struct Mol Biol ; 26(1): 25-34, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598552

RESUMO

The functionally tolerated sequence space of proteins can now be explored in an unprecedented way, owing to the expansion of genomic databases and the development of high-throughput methods to interrogate protein function. For signaling proteins, several recent studies have shown how the analysis of sequence variation leverages the available protein-structure information to provide new insights into specificity and allosteric regulation. In this Review, we discuss recent work that illustrates how this emerging approach is providing a deeper understanding of signaling proteins.


Assuntos
Proteínas/química , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Modelos Biológicos , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Proteínas/genética , Transdução de Sinais/genética , Especificidade por Substrato
19.
Crit Rev Biochem Mol Biol ; 53(5): 535-563, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30183386

RESUMO

Tyrosine kinases were first discovered as the protein products of viral oncogenes. We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members. Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals. Several families of cytoplasmic tyrosine kinases have in common a core architecture, the "Src module," composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain. Each of these families is defined by additional elaborations on this core architecture. Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module. The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.


Assuntos
Citoplasma/enzimologia , Quinases da Família src/química , Quinases da Família src/metabolismo , Regulação Alostérica , Animais , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Evolução Molecular , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Especificidade por Substrato , Domínios de Homologia de src , Quinases da Família src/genética
20.
Proc Natl Acad Sci U S A ; 115(31): E7303-E7312, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30012625

RESUMO

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as "phosphosites") must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the -1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.


Assuntos
Receptores ErbB/química , Análise Mutacional de DNA , Receptores ErbB/fisiologia , Humanos , Fosforilação , Conformação Proteica , Proteoma , Transdução de Sinais/fisiologia
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