RESUMEN
Intrinsically disordered regions in proteins often function as binding motifs in protein-protein interactions. The mechanistic aspects and molecular details of such coupled binding and folding reactions, which involve formation of multiple noncovalent bonds, have been broadly studied theoretically, but experimental data are scarce. Here, using a combination of protein semisynthesis to incorporate phosphorylated amino acids, backbone amide-to-ester modifications, side chain substitutions, and binding kinetics, we examined the interaction between the intrinsically disordered motif of amyloid precursor protein (APP) and the phosphotyrosine binding (PTB) domain of Mint2. We show that the interaction is regulated by a self-inhibitory segment of the PTB domain previously termed ARM. The helical ARM linker decreases the association rate constant 30-fold through a fast pre-equilibrium between an open and a closed state. Extensive side chain substitutions combined with kinetic experiments demonstrate that the rate-limiting transition state for the binding reaction is governed by native and non-native hydrophobic interactions and hydrogen bonds. Hydrophobic interactions were found to be particularly important during crossing of the transition state barrier. Furthermore, linear free energy relationships show that the overall coupled binding and folding reaction involves cooperative formation of interactions with roughly 30% native contacts formed at the transition state. Our data support an emerging picture of coupled binding and folding reactions following overall chemical principles similar to those of folding of globular protein domains but with greater malleability of ground and transition states.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Cadherinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Precursor de Proteína beta-Amiloide/síntesis química , Precursor de Proteína beta-Amiloide/genética , Animales , Cadherinas/síntesis química , Cadherinas/genética , Proteínas Portadoras/síntesis química , Proteínas Portadoras/genética , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Intrínsecamente Desordenadas/síntesis química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Cinética , Mutación , Proteínas del Tejido Nervioso/síntesis química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Dominios Proteicos/genética , Ingeniería de Proteínas , Pliegue de Proteína , Ratas , TermodinámicaRESUMEN
E-cadherin-mediated cell-cell interactions in the zonula adherens play an important role in the formation of the intercellular tight junctions found in the blood-brain barrier. However, it is also responsible for the low permeation of drugs into the brain. In this study, HAV6 peptide derived from the EC1 domain of E-cadherin was found to enhance the permeation of ¹4C-mannitol and [³H(G)]-daunomycin through the blood-brain barrier of the in situ rat brain perfusion model. In addition, HAV6 peptide and verapamil have a synergistic effect in enhancing the BBB permeation of daunomycin. A new intercellular-junction resealing assay was also developed using Caco-2 monolayers to evaluate new peptides (BLG2, BLG3, and BLG4) derived from the bulge regions of the EC2, EC3, and EC4 domains of E-cadherin. BLG2 and BLG4 peptides but not BLG3 peptides were found to be effective in blocking the resealing of the intercellular junctions. The positive control peptides (ADT10, ADT6, and HAV10) block the resealing of the intercellular junctions in a concentration-dependent manner. All these findings suggest that E-cadherin-derived peptides can block E-cadherin-mediated cell-cell interactions. These findings demonstrate that cadherin peptides may offer a useful targeted permeation enhancement of therapeutic agents such as anticancer drugs into the brain.
