Tanaka Naoki, Tani Yasushi, Hattori Hiroyuki, Tada Tomoko, Kunugi Shigeru
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan.
Protein Sci. 2004 Dec;13(12):3214-21. doi: 10.1110/ps.04780704. Epub 2004 Nov 10.
The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.
大肠杆菌热休克蛋白ClpB与DnaK伴侣系统协同作用，使蛋白质聚集体重新激活。ClpB的N端结构域在伴侣活性中起重要作用，但其机制尚不清楚。在本研究中，我们研究了ClpB N端结构域对苹果酸脱氢酶（MDH）重折叠的影响。ClpB通过与中间体的强烈相互作用降低了MDH重折叠的产率。然而，ClpB N端结构域缺失（ClpBDeltaN）对重折叠动力学没有影响，这表明MDH重折叠受到与N端结构域相互作用的影响。此外，在ClpB N端片段（ClpBN）存在的情况下，MDH重折叠产率提高了50%。荧光偏振分析表明，这种类似伴侣的活性最好由ClpBN与MDH可逆聚集体之间的弱相互作用来解释。根据重折叠动力学计算，ClpBN与可逆聚集体的解离常数估计为45μM。ClpBN表面Leu 97和Leu 110处的氨基酸取代降低了类似伴侣的活性和对底物的亲和力。此外，这些残基参与了ClpB中ATPase活性的刺激。因此，Leu 97和Leu 110负责底物识别和ATP诱导的ClpB构象变化的调节。
