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Tailoring echistatin to possess higher affinity for integrin α IIb β 3
Author(s) -
Yamada Takao,
Kidera Akinori
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00409-7
Subject(s) - disintegrin , integrin , mutant , chemistry , peptide , folding (dsp implementation) , stereochemistry , biophysics , receptor , biochemistry , biology , metalloproteinase , enzyme , electrical engineering , gene , engineering
A mutant of echistatin, a disintegrin with a high affinity for the integrins, was constructed by substituting RGD for RGD in the Arg‐Gly‐Asp (RGD) region. The mutant was chemically synthesized, subjected to a folding process with air oxidation, and purified by reverse‐phase HPLC. The peptide mapping and mass spectrometric analyses revealed that the two Cys residues introduced in the mutant are linked to each other, without any effect on the mode of the four disulfide bonds present in native echistatin, as expected. The mutant strongly inhibited the binding of human fibrinogen to its receptor, integrin α IIb β 3 , with an IC 50 value of 0.12 nM. This value shows that the mutant is twice as potent as the native form (IC 50 = 0.23 nM). These results indicate that the native disintegrin molecule, which has been considered to possess the optimum affinity for the integrins, can be tailored to exhibit even higher affinity by introducing the conformational constraint into the RGD region. Monte Carlo simulations of KRCRGDCMD, the RGD region in the mutant, suggested that the disulfide bond constrains the RGD region to assume a type II′ β‐turn, with Gly and Asp in positions 2 and 3 of the turn.