Maity Mritunjoy, Dolui Sandip, Maiti Nakul C
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India.
Phys Chem Chem Phys. 2015 Dec 14;17(46):31216-27. doi: 10.1039/c5cp04661k.
An analog of coomassie brilliant blue-R (CBB-R) was recently found to act as an antagonist to ATP-sensitive purinergic receptors (P2X7R) and has potential to be used in medicine. With the aim of understanding its transportation and distribution through blood, in this investigation, we measured the binding parameters of CBB-R with bovine hemoglobin (BHG). The molecule specifically bound to a single binding site of the protein with a stoichiometric ratio of 1 : 1 and the observed binding constant Ka was 3.5, 2.5, 2.0 and 1.5 × 10(5) M(-1) at 20 °C, 27 °C, 37 °C and 45 °C, respectively. The measured respective ΔG(0) values of the binding at four temperatures were -30.45, -22.44, -18.04 and -11.95 kJ mol(-1). The ΔH(0) (change in enthalpy) and ΔS(0) (change in entropy) values were -23.6 kJ mol(-1) and -70.66 J mol(-1) respectively in the binding process. The negative value of ΔH(0) and ΔS(0) indicated that the binding of the molecule was thermodynamically favorable. The best energy structure in the molecular docking analysis revealed that CBB-R preferred to be intercalated in the cavity among the α2, β1 and β2 subunits and the binding location was 7.4 Å away from Trp37 in the β2 subunit. The binding of the molecule with the protein was stabilized by hydrogen bonds involving the side chain of two amino acid residues. The residues were Lys104 and Glu101 in the β2 subunit. The binding was further stabilized via hydrogen bond formation between the amide group of the peptide backbone (residue Tyr145 of the β1 subunit) and CBB-R. A shift of the amide I (-C=O stretching) band frequency of ∼8 cm(-1) to low energy was ascribed to the hydrogen bond interaction involving the polypeptide carbonyl of the protein and the CBB-R molecule. In addition, two π-cation interactions between Lys99 of the α2 subunit and Lys104 of the β2 subunit and CBB-R contributed favorably in the binding processes. No substantial change in the soret and Q absorption bands of BHG could be observed in the presence of CBB-R. It indicated that the oxygen binding domain or the heme proximity was not blocked or substantially perturbed due to the binding of CBB-R. The circular dichroism and the molecular dynamics analysis further established that the binding interaction caused no significant alteration in the protein long range secondary structure.
最近发现考马斯亮蓝-R（CBB-R）的一种类似物可作为ATP敏感性嘌呤能受体（P2X7R）的拮抗剂，具有医学应用潜力。为了解其在血液中的转运和分布情况，在本研究中，我们测量了CBB-R与牛血红蛋白（BHG）的结合参数。该分子特异性结合到蛋白质的单个结合位点，化学计量比为1∶1，在20℃、27℃、37℃和45℃下观察到的结合常数Ka分别为3.5、2.5、2.0和1.5×10⁵ M⁻¹。在四个温度下测得的结合反应各自的ΔG⁰值分别为-30.45、-22.44、-18.04和-11.95 kJ·mol⁻¹。结合过程中的ΔH⁰（焓变）和ΔS⁰（熵变）值分别为-23.6 kJ·mol⁻¹和-70.66 J·mol⁻¹·K⁻¹。ΔH⁰和ΔS⁰的负值表明该分子的结合在热力学上是有利的。分子对接分析中的最佳能量结构显示，CBB-R更倾向于插入α2、β1和β2亚基之间的腔中，结合位置距离β2亚基中的Trp37为7.4 Å。该分子与蛋白质的结合通过涉及两个氨基酸残基侧链的氢键得以稳定。这些残基是β2亚基中的Lys104和Glu101。通过肽主链的酰胺基团（β1亚基的Tyr145残基）与CBB-R之间形成氢键，结合进一步得到稳定。酰胺I（-C=O伸缩）带频率向低能量方向移动约8 cm⁻¹归因于蛋白质的多肽羰基与CBB-R分子之间的氢键相互作用。此外，α2亚基的Lys99、β2亚基的Lys104与CBB-R之间的两个π-阳离子相互作用对结合过程有有利贡献。在CBB-R存在的情况下，未观察到BHG的索雷特带和Q吸收带发生实质性变化。这表明由于CBB-R的结合，氧结合域或血红素附近未被阻断或受到实质性扰动。圆二色性和分子动力学分析进一步证实，这种结合相互作用未引起蛋白质远程二级结构的显著改变。
