Takenaka Toshio, Nakamura Takashi, Yanaka Saeko, Yagi-Utsumi Maho, Chandak Mahesh S, Takahashi Kazunobu, Paul Subhankar, Makabe Koki, Arai Munehito, Kato Koichi, Kuwajima Kunihiro
Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi, Japan.
Department of Functional Molecular Science, School of Physical Sciences, the Graduate University for Advanced Studies (Sokendai), Myodaiji, Okazaki, Aichi, Japan.
PLoS One. 2017 Oct 23;12(10):e0187022. doi: 10.1371/journal.pone.0187022. eCollection 2017.
We studied the interaction between GroES and a single-ring mutant (SR1) of GroEL by the NMR titration of 15N-labeled GroES with SR1 at three different temperatures (20, 25 and 30°C) in the presence of 3 mM ADP in 100 mM KCl and 10 mM MgCl2 at pH 7.5. We used SR1 instead of wild-type double-ring GroEL to precisely control the stoichiometry of the GroES binding to be 1:1 ([SR1]:[GroES]). Native heptameric GroES was very flexible, showing well resolved cross peaks of the residues in a mobile loop segment (residue 17-34) and at the top of a roof hairpin (Asn51) in the heteronuclear single quantum coherence spectra. The binding of SR1 to GroES caused the cross peaks to disappear simultaneously, and hence it occurred in a single-step cooperative manner with significant immobilization of the whole GroES structure. The binding was thus entropic with a positive entropy change (219 J/mol/K) and a positive enthalpy change (35 kJ/mol), and the binding constant was estimated at 1.9×105 M-1 at 25°C. The NMR titration in 3 mM ATP also indicated that the binding constant between GroES and SR1 increased more than tenfold as compared with the binding constant in 3 mM ADP. These results will be discussed in relation to the structure and mechanisms of the chaperonin GroEL/GroES complex.
我们在pH 7.5、100 mM KCl和10 mM MgCl₂条件下，于三种不同温度（20、25和30°C）下，通过用SR1对15N标记的GroES进行NMR滴定，研究了GroES与GroEL的单环突变体（SR1）之间的相互作用，体系中存在3 mM ADP。我们使用SR1而非野生型双环GroEL，以精确控制GroES结合的化学计量比为1:1（[SR1]:[GroES]）。天然七聚体GroES非常灵活，在异核单量子相干谱中，其移动环段（残基17 - 34）和屋顶发夹顶部（Asn51）的残基显示出分辨率良好的交叉峰。SR1与GroES的结合导致交叉峰同时消失，因此它以单步协同方式发生，伴随着整个GroES结构的显著固定。这种结合是熵驱动的，熵变正值为219 J/mol/K，焓变正值为35 kJ/mol，25°C时结合常数估计为1.9×10⁵ M⁻¹。在3 mM ATP条件下的NMR滴定还表明，与3 mM ADP条件下的结合常数相比，GroES与SR1之间的结合常数增加了十多倍。这些结果将结合伴侣蛋白GroEL/GroES复合物的结构和机制进行讨论。
