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1.
Structure ; 28(1): 54-62.e5, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31780432

RESUMO

Epidermal growth factor receptors (EGFRs) are central cellular signaling interfaces whose misregulation is related to several severe diseases. Although ligand binding to the extracellular domain is the most obvious regulatory element, also intracellular factors can act as modulators of EGFR activity. The juxtamembrane (JM) segment seems to be the receptor's key interaction interface of these cytoplasmic factors. However, only a limited number of cytoplasmic EGFR modulators are known and a comprehensive understanding of their mode of action is lacking. Here, we report ARNO, a member of the cytohesin family, as another JM-binding protein and structurally characterize the ARNO-EGFR interaction interface. We reveal that its binding mode displays common features and distinct differences with JM's interaction with calmodulin and anionic phospholipids. Furthermore, we show that each interaction can be modulated by additional factors, generating a distinctly regulated network of possible EGFR modulators acting on the intracellular domain of the receptor.


Assuntos
Calmodulina/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fosfolipídeos/metabolismo , Sítios de Ligação , Citoplasma/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Proteínas Ativadoras de GTPase/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
2.
Chemistry ; 24(26): 6665-6671, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29369431

RESUMO

Pulsed electron-electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface-exposed cysteine residues, which can be efficiently spin-labelled using thiol-reactive nitroxide compounds. This method has been coined "site-directed spin labelling" (SDSL) and is normally straight-forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of "inhibitor-directed spin labelling" (IDSL) as an approach to spin label suitable cysteine-rich proteins in a site-directed and highly specific manner by employing bespoke spin-labelled inhibitors. Advantages and disadvantages of IDSL are discussed.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Proteínas/química , Óxidos N-Cíclicos/química , Cisteína/química , Receptores ErbB/química , Receptores ErbB/metabolismo , Mesilatos/química , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas/metabolismo , Soluções/química , Marcadores de Spin
3.
Angew Chem Int Ed Engl ; 56(29): 8417-8421, 2017 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-28628261

RESUMO

The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near-full-length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof-of-concept that inhibitor-based spin labeling enables the convenient introduction of site-specific spin labels into kinases for which covalent or tight-binding small-molecule modulators are available.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinazolinas/síntese química , Quinazolinas/química , Soluções , Relação Estrutura-Atividade
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