Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Office of Intramural Research, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 2019 May 31;431(12):2331-2342. doi: 10.1016/j.jmb.2019.04.040. Epub 2019 May 6.
There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS) and one for nitrogen regulation (PTS), that utilize proteins enzyme I (EI) and HPr, and enzyme I (EI) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.
大肠杆菌中有两个旁系同源的磷酸转移酶系统，一个用于糖摄取（PTS），另一个用于氮调控（PTS），它们分别利用酶 I（EI）和 HPr 以及酶 I（EI）和 NPr。酶 I 蛋白具有相似的折叠结构，其底物 HPr 和 NPr 也是如此，但它们在立体特异性蛋白-蛋白复合物形成和可逆磷酸转移中对其同源伴侣表现出严格的特异性。在这里，我们通过研究瞬时相遇复合物来研究特定 EI:NPr 复合物形成的机制。NMR 顺磁松弛增强实验表明，EI 不仅与预期的伴侣 NPr 形成瞬时相遇复合物，还与意外的伴侣 HPr 形成瞬时相遇复合物。HPr 占据 EI 上的非生产性瞬时位点，但无法完成立体特异性复合物形成。通过占据非生产性瞬时位点，HPr 促进 NPr 瞬时相互作用到更接近立体特异性结合位点的生产性位点，并实际上增强了 NPr 和 EI 之间的特异性复合形成。HPr 的细胞水平大约是 NPr 的 150 倍。因此，我们的发现表明，通过形成竞争性相遇复合物，可能存在一种交叉调节酶活性的机制。
