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 containing 65P, 65PR, 65PRYH, 65PRNGLYG, and 65PRNPWNG C-terminal sequences. The effect of C-terminal region on their integrin binding affinities was αIIbβ3 > αvβ3 ≥ α5β1, and the 48ARGDWN-65PRNPWNG protein was the most selective integrin αIIbβ3 mutant. The 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG mutants had similar activities in inhibiting platelet aggregation and the binding of fibrinogen to platelet. In contrast, 48ARGDWN-65PRYH and 48ARGDWN-65PRNGLYG exhibited 2.9- and 3.0-fold decreases in inhibiting cell adhesion in comparison with that of 48ARGDWN-65PRNPWNG. 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 48ARGDWN-65PRYH, 48ARGDWN-65PRNGLYG, and 48ARGDWN-65PRNPWNG 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-65PRNPWNG 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.
Highlights
RGD-containing disintegrins are potent integrin inhibitors that were found in snake venoms [1,2,3,4]
The docking of the 48ARGDWN-65PRNPWNG 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
Short disintegrins are composed of 41 to 51 residues and four disulfide bonds; medium disintegrins contain approximately 70 amino acids and six disulfide bonds; long disintegrins include a polypeptide with approximately 84 residues cross-linked by seven disulfide bonds; and homo- and hetero-dimeric disintegrins contain each subunit of approximately 67 residues with a total of ten disulfide bonds involved in the formation of four intrachain disulfides and two interchain disulfides [6]
Summary
RGD-containing disintegrins are potent integrin inhibitors that were found in snake venoms [1,2,3,4]. They are classified into small, medium, long, and dimeric disintegrins based on their size and the number of disulfide bonds [5]. The pairing of cysteine residues in disintegrins play an important role in exposing the RGD binding motif that mediates inhibition of platelet aggregation, neutrophil or endothelial cell function [1,2,3,4,5,6,7]. Disintegrins are used to develop anti-platelets agents and anti-angiogenesis inhibitors for cancer [1,2,3,4,5,6]
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