RESUMO
The endoplasmic reticulum malectin is a highly conserved protein in the animal kingdom that has no counterpart so far in lower organisms. We recently determined the structure of its conserved domain and found a highly selective binding to Glc(2)Man(9)GlcNAc(2), an intermediate of N-glycosylation. In our quest for putative ligands during the initial characterization of the protein, we noticed that the malectin domain is highly specific for diglucosides but quite tolerant towards the linkage of the glucosidic bond. To understand the molecular requirements for the observed promiscuity of the malectin domain, here we analyze the binding to a range of diglucosides through comparison of the protein chemical shift perturbation patterns and the saturation transfer difference spectra of the ligands including two maltose-mimicking drugs. A comparison of the maltose-bound structure of the malectin domain with the complex of the native ligand nigerose reveals why malectin is able to tolerate such a diversity of ligands.
Assuntos
Glucosídeos/química , Lectinas/química , Proteínas de Membrana/química , Animais , Retículo Endoplasmático/química , Lectinas/isolamento & purificação , Espectroscopia de Ressonância Magnética/normas , Proteínas de Membrana/isolamento & purificação , Modelos Moleculares , Estrutura Terciária de Proteína , Padrões de Referência , Xenopus laevisRESUMO
The family of cysteine rich proteins (CRP) comprises three closely homologous members that have been reported to interact with alpha-actinin. Muscular LIM protein (MLP/CRP3), the skeletal muscle variant, was originally discovered as a positive regulator of myogenesis and is suggested to be part of the stretch sensor of the myofibril through its interaction with telethonin (T-Cap). We determined the structure of both LIM domains of human MLP by nuclear magnetic resonance spectroscopy. We confirm by (15)N relaxation measurements that both LIM domains act as independent units and that the adjacent linker regions are fully flexible. With the published structures of CRP1 and CRP2, the complete family has now been structurally characterized.
Assuntos
Proteínas Musculares/química , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Aviárias/química , Proteínas de Transporte/química , Proteínas de Ligação a DNA/química , Proteínas do Olho/química , Proteínas de Homeodomínio/química , Humanos , Proteínas com Domínio LIM , Proteínas com Homeodomínio LIM , Proteínas de Membrana , Modelos Moleculares , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Termodinâmica , Fatores de TranscriçãoRESUMO
N-Glycosylation starts in the endoplasmic reticulum (ER) where a 14-sugar glycan composed of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc(3)Man(9)GlcNAc(2)) is transferred to nascent proteins. The glucoses are sequentially trimmed by ER-resident glucosidases. The Glc(3)Man(9)GlcNAc(2) moiety is the substrate for oligosaccharyltransferase; the Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) intermediates are signals for glycoprotein folding and quality control in the calnexin/calreticulin cycle. Here, we report a novel membrane-anchored ER protein that is highly conserved in animals and that recognizes the Glc(2)-N-glycan. Structure determination by nuclear magnetic resonance showed that its luminal part is a carbohydrate binding domain that recognizes glucose oligomers. Carbohydrate microarray analyses revealed a uniquely selective binding to a Glc(2)-N-glycan probe. The localization, structure, and binding specificity of this protein, which we have named malectin, open the way to studies of its role in the genesis, processing and secretion of N-glycosylated proteins.
Assuntos
Carboidratos/química , Retículo Endoplasmático/metabolismo , Lectinas/fisiologia , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Animais , Calnexina/química , Calreticulina/química , Linhagem Celular Tumoral , Glicosilação , Humanos , Lectinas/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
The family of CRP proteins comprises three members, which are composed of two LIM domains separated by a long linker of more than 50 residues. We determined the structure of the muscle variant, MLP (CRP3), by nuclear magnetic resonance and show that the two LIM domains are independent of each other.
