Decorsin,an antagonist of integrin glycoprotein IIb/IIIa,contains Arg-Gly-Asp(RGD)sequence and three disulfide bridges.The function of RGD sequence has already been well defined,but the roles of conserved disulfide bonds in antihemostatic proteins still remain unclear.Herein we use the fusion expression and characterization of mutant decorsin to study the functions of disulfide bonds in protein structure,stability and biological activity.The purified protein shows an apparent inhibition of activity to platelet aggregation induced by ADP with IC50 of 500 nM.The removal of cys7-cys15(from cysteine to serine)at the N-terminal causes a thirty-fold decrease of the inhibition activity with IC50 of 15 M,whereas the mutation of cys22-cys38 at the C-terminal completely impairs the biological activity of decorsin.The overall secondary and tertiary structures of decorsin are disrupted inevitably without disulfide bonds.Using a domain insertion mutation,the retaining of RGD loop and the adjacent disulfide bond produces a week antihemostatic activity of decorsin.This reveals that the overall structure of decorsin stabilized by the three conserved disulfide bridges is cooperative for antihemostatic function.Our study on the effect of disulfide bonds together with RGD-sequence on the protein function is helpful for structure-based drug design of antithrombotic research.
The protein-based molecular recognition of the adenine ring is essential to understand protein function and drug design as well.In this paper,a variety of the adenine-based inhibitors with modified groups of amino groups,nitrogen and oxygen atoms in the aromatic ring are designed,and the binding capability of these adenosine analogues with an aminoglycoside antibiotic kinase [APH(3')-IIIa] are investigated with activity assays and isothermal titration calorimetry(ITC) experiments.1-aminoisoquinoline is one of the weakest substrates bound to APH(3')-IIIa with the lowest affinity(high ki and high kd) and the smallest negative value of free energy change(G) among the inhibitors tested.The binding process of adenine and 5-nitroisoquinoline to APH(3')-IIIa is an enthalpy-driven event with unfavorable entropy,which is consistent with the energy change induced by the binding of ATP to the enzyme.However,the reverse is true for 1-aminoisoquinoline,3-amino-5-nitrobenzisothiazole,5-aminoisoquinoline binding to the enzyme because the entropy is more favorable and the enthalpy makes a lower contribution to the binding process.These results suggest that small changes of the adenine ring can lead to significant influence on the ability of these analogues to occupy the adenine-binding region of the enzyme,which can be the potential inhibitors as drug candidates against the bacterial resistance.
