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

Yao Tsung Chang, Jia Hau Shiu, Chun Hao Huang, Yi Chun Chen, Chiu Yueh Chen, Yung Sheng Chang, Woei Jer Chuang

Research output: Contribution to journalArticle

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Abstract

Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP 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 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 o>α5β1, and the48ARGDWN- 48ARGDWN-56PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-56PRYH and 48ARGDWN- 48ARGDWN-56PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of48ARGDWN- 48ARGDWN-56PRNPWNG. 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 of48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN- 48ARGDWN-56PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-? 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.

Original languageEnglish
Article numbere0175321
JournalPloS one
Volume12
Issue number4
DOIs
Publication statusPublished - 2017 Apr

Fingerprint

integrins
Integrins
Platelets
Disintegrins
mutants
fibrinogen
Fibrinogen
platelet aggregation
Cell adhesion
Platelet Aggregation
cell adhesion
Cell Adhesion
Blood Platelets
Agglomeration
Pichia pastoris
Pichia
hydrogen
rhodostomin
Cations
Hydrogen

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

@article{f190595cdac54c8d9bd164bbd6504df8,
title = "Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition",
abstract = "Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP 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 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 o>α5β1, and the48ARGDWN- 48ARGDWN-56PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-56PRYH and 48ARGDWN- 48ARGDWN-56PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of48ARGDWN- 48ARGDWN-56PRNPWNG. 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 of48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN- 48ARGDWN-56PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-? 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.",
author = "Chang, {Yao Tsung} and Shiu, {Jia Hau} and Huang, {Chun Hao} and Chen, {Yi Chun} and Chen, {Chiu Yueh} and Chang, {Yung Sheng} and Chuang, {Woei Jer}",
year = "2017",
month = "4",
doi = "10.1371/journal.pone.0175321",
language = "English",
volume = "12",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "4",

}

Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition. / Chang, Yao Tsung; Shiu, Jia Hau; Huang, Chun Hao; Chen, Yi Chun; Chen, Chiu Yueh; Chang, Yung Sheng; Chuang, Woei Jer.

In: PloS one, Vol. 12, No. 4, e0175321, 04.2017.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

AU - Chang, Yao Tsung

AU - Shiu, Jia Hau

AU - Huang, Chun Hao

AU - Chen, Yi Chun

AU - Chen, Chiu Yueh

AU - Chang, Yung Sheng

AU - Chuang, Woei Jer

PY - 2017/4

Y1 - 2017/4

N2 - Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP 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 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 o>α5β1, and the48ARGDWN- 48ARGDWN-56PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-56PRYH and 48ARGDWN- 48ARGDWN-56PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of48ARGDWN- 48ARGDWN-56PRNPWNG. 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 of48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN- 48ARGDWN-56PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-? 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.

AB - Rhodostomin (Rho) is a medium disintegrin containing a 48PRGDMP 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 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 o>α5β1, and the48ARGDWN- 48ARGDWN-56PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-56PRYH and 48ARGDWN- 48ARGDWN-56PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of48ARGDWN- 48ARGDWN-56PRNPWNG. 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 of48ARGDWN- 48ARGDWN-56PRYH, 48ARGDWN- 48ARGDWN-56PRNGLYG, and 48ARGDWN- 48ARGDWN-56PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop. In particular, the W52 sidechain of 48ARGDWN interacted with H68 of 65PRYH, L69 of 65PRNGLYG, and N70 of 65PRNPWNG, respectively. The docking of the 48ARGDWN- 48ARGDWN-56PRNPWNG mutant into integrin αIIbβ3 showed that the N70 residue formed hydrogen bonds with the αIIb D159 residue, and the W69 residue formed cation-? 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.

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DO - 10.1371/journal.pone.0175321

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