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1.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 11): 697-706, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31702583

RESUMO

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab-benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.


Assuntos
Anticorpos/química , Cocaína/imunologia , Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Cocaína/análogos & derivados , Cocaína/química , Cristalização , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Ligantes , Domínios Proteicos , Proteínas Recombinantes/química
2.
J Biol Chem ; 292(25): 10549-10563, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28487372

RESUMO

The Notch pathway is a cell-to-cell signaling mechanism that is essential for tissue development and maintenance, and aberrant Notch signaling has been implicated in various cancers, congenital defects, and cardiovascular diseases. Notch signaling activates the expression of target genes, which are regulated by the transcription factor CSL (CBF1/RBP-J, Su(H), Lag-1). CSL interacts with both transcriptional corepressor and coactivator proteins, functioning as both a repressor and activator, respectively. Although Notch activation complexes are relatively well understood at the structural level, less is known about how CSL interacts with corepressors. Recently, a new RBP-J (mammalian CSL ortholog)-interacting protein termed RITA has been identified and shown to export RBP-J out of the nucleus, thereby leading to the down-regulation of Notch target gene expression. However, the molecular details of RBP-J/RITA interactions are unclear. Here, using a combination of biochemical/cellular, structural, and biophysical techniques, we demonstrate that endogenous RBP-J and RITA proteins interact in cells, map the binding regions necessary for RBP-J·RITA complex formation, and determine the X-ray structure of the RBP-J·RITA complex bound to DNA. To validate the structure and glean more insights into function, we tested structure-based RBP-J and RITA mutants with biochemical/cellular assays and isothermal titration calorimetry. Whereas our structural and biophysical studies demonstrate that RITA binds RBP-J similarly to the RAM (RBP-J-associated molecule) domain of Notch, our biochemical and cellular assays suggest that RITA interacts with additional regions in RBP-J. Taken together, these results provide molecular insights into the mechanism of RITA-mediated regulation of Notch signaling, contributing to our understanding of how CSL functions as a transcriptional repressor of Notch target genes.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Células A549 , Animais , Cristalografia por Raios X , DNA , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas de Neoplasias/genética , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade
3.
PLoS Biol ; 14(7): e1002509, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27404588

RESUMO

Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.


Assuntos
Proteínas de Drosophila/química , Drosophila melanogaster , Proteínas Repressoras/química , Fatores de Transcrição/química , Animais , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , DNA/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Nucleic Acids Res ; 44(10): 4703-20, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26912830

RESUMO

The transcriptional shift from repression to activation of target genes is crucial for the fidelity of Notch responses through incompletely understood mechanisms that likely involve chromatin-based control. To activate silenced genes, repressive chromatin marks are removed and active marks must be acquired. Histone H3 lysine-4 (H3K4) demethylases are key chromatin modifiers that establish the repressive chromatin state at Notch target genes. However, the counteracting histone methyltransferase required for the active chromatin state remained elusive. Here, we show that the RBP-J interacting factor SHARP is not only able to interact with the NCoR corepressor complex, but also with the H3K4 methyltransferase KMT2D coactivator complex. KMT2D and NCoR compete for the C-terminal SPOC-domain of SHARP. We reveal that the SPOC-domain exclusively binds to phosphorylated NCoR. The balance between NCoR and KMT2D binding is shifted upon mutating the phosphorylation sites of NCoR or upon inhibition of the NCoR kinase CK2ß. Furthermore, we show that the homologs of SHARP and KMT2D in Drosophila also physically interact and control Notch-mediated functions in vivo Together, our findings reveal how signaling can fine-tune a committed chromatin state by phosphorylation of a pivotal chromatin-modifier.


Assuntos
Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Regulação da Expressão Gênica , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transcrição Gênica , Animais , Caseína Quinase II/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Proteínas Nucleares/química , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA , Xenopus laevis
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