Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

Rhodostomin (Rho) is a medium disintegrin containing a (48)PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho (48)PRGDMP and (48)ARGDWN...

Descripción completa

Detalles Bibliográficos
Autores principales: Chang, Yao-Tsung, Shiu, Jia-Hau, Huang, Chun-Hao, Chen, Yi-Chun, Chen, Chiu-Yueh, Chang, Yung-Sheng, Chuang, Woei-Jer
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Research Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388508/
https://www.ncbi.nlm.nih.gov/pubmed/28399159
http://dx.doi.org/10.1371/journal.pone.0175321
Descripción
Sumario:Rhodostomin (Rho) is a medium disintegrin containing a (48)PRGDMP motif. We here showed that Rho proteins with P48A, M52W, and P53N mutations can selectively inhibit integrin αIIbβ3. To study the roles of the RGD loop and C-terminal region in disintegrins, we expressed Rho (48)PRGDMP and (48)ARGDWN mutants in Pichia pastoris containing (65)P, (65)PR, (65)PRYH, (65)PRNGLYG, and (65)PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the (48)ARGDWN-(65)PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The (48)ARGDWN-(65)PRYH, (48)ARGDWN-(65)PRNGLYG, and (48)ARGDWN-(65)PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, (48)ARGDWN-(65)PRYH and (48)ARGDWN-(65)PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of (48)ARGDWN-(65)PRNPWNG. Based on the results of cell adhesion, platelet aggregation and the binding of fibrinogen to platelet inhibited by ARGDWN mutants, integrin αIIbβ3 bound differently to immobilized and soluble fibrinogen. NMR structural analyses of (48)ARGDWN-(65)PRYH, (48)ARGDWN-(65)PRNGLYG, and (48)ARGDWN-(65)PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of (48)ARGDWN interacted with H68 of (65)PRYH, L69 of (65)PRNGLYG, and N70 of (65)PRNPWNG, respectively. The docking of the (48)ARGDWN-(65)PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-π interaction with the β3 K125 residue. These results provide the first structural evidence that the interactions between the RGD loop and C-terminus of medium disintegrins depend on their amino acid sequences, resulting in their functional differences in the binding and selectivity of integrins.

Ejemplares similares