RESUMO
Chitin-binding domain of chitinase A1 (ChBDChiA1 ) is characteristic because it binds only to insoluble crystalline chitin. While binding sites of major carbohydrate-binding modules carry multiple aromatic rings aligned on a surface, lethal mutations for ChBDChiA1 were reported only at W687, a location completely different from the site mentioned above, in spite of their similar main-chain folds. Here, the structural mechanism underlying its crystalline chitin binding was uncovered by solid-state NMR. Based on 13 C- and 15 N-signal assignment of microcrystalline ChBDChiA1 , the chemical shift perturbation on chitin binding was carefully examined. The perturbation was greatest at W687 and nonaromatic residues surrounding it, revealing their direct involvement in chitin binding. These residues and Q679 should provide a novel chitin-binding platform parallel to the W687 ring.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Quitina/química , Quitinases/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Sequência de Carboidratos , Quitina/metabolismo , Quitinases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios ProteicosRESUMO
Bin1/M-amphiphysin-II is an amphiphysin-II isoform highly expressed in transverse tubules of adult striated muscle and is implicated in their biogenesis. Bin1 contains a basic unique amino-acid sequence, Exon10, which interacts with certain phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), to localize to membranes. Here we found that Exon10 also binds to the src homology 3 (SH3) domain of Bin1 itself, and hence blocks the binding of the SH3 domain to its canonical PxxP ligands, including dynamin. This blockage was released by addition of PI(4,5)P(2) in vitro or in cells overexpressing phosphatidylinositol 4-phosphate 5-kinase. The Exon10-binding interface of the Bin1 SH3 domain largely overlapped with its PxxP-binding interface. We also show that the PLCdelta pleckstrin homology domain, another PI(4,5)P(2)-binding module, cannot substitute for Exon10 in Bin1 function in transverse tubule formation, and suggest the importance of the dual biochemical properties of Exon10 in myogenesis. Our results exemplify a novel mechanism of SH3 domain regulation, and suggest that the SH3-mediated protein-protein interactions of Bin1 are regulated by Exon10 so that it may only occur when Bin1 localizes to certain submembrane areas.
