RESUMEN
Interactions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14-3-3 adapter proteins are a prominent example and typically bind partner proteins in a phosphorylation-dependent mono- or bivalent manner. Herein we describe the development of a cucurbit[8]uril (Q8)-based supramolecular system, which in conjunction with the 14-3-3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine-glycine-glycine (FGG) tripeptide motif to the N-terminus of the 14-3-3-binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8-induced dimerization of the ERα epitope augmented its affinity towards 14-3-3 through a binary bivalent binding mode. The crystal structure of the Q8-induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8.
Asunto(s)
Proteínas 14-3-3/química , Hidrocarburos Aromáticos con Puentes/química , Imidazoles/química , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Unión Competitiva , Hidrocarburos Aromáticos con Puentes/metabolismo , Cristalografía por Rayos X , Dimerización , Epítopos/química , Epítopos/metabolismo , Colorantes Fluorescentes/química , Fluorometría , Imidazoles/metabolismo , Simulación de Dinámica Molecular , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for inâ vitro supramolecular signaling networks.