ATP-Induced Dimerization of the F0F1 蔚 Subunit from Bacillus PS3: A Hydrogen Exchange鈥揗ass Spectrometry Study

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• 作者：Antony D. Rodriguez ; Stanley D. Dunn ; Lars Konermann
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：June 24, 2014
• 年：2014
• 卷：53
• 期：24
• 页码：4072-4080
• 全文大小：508K
• ISSN：1520-4995

文摘

F₀F₁ ATP synthase harnesses a transmembrane electrochemical gradient for the production of ATP. When operated in reverse, this multiprotein complex catalyzes ATP hydrolysis. In bacteria, the 蔚 subunit is involved in regulating this ATPase activity. Also, 蔚 is essential for coupling ATP hydrolysis (or synthesis) to proton translocation. The 蔚 subunit consists of a 尾 sandwich and two C-terminal helices, 伪1 and 伪2. The protein can switch from a compact fold to an alternate conformation where 伪1 and 伪2 are separated, resulting in an extended structure. 蔚 from the thermophile Bacillus PS3 (T蔚) binds ATP with high affinity such that this protein may function as an intracellular ATP level sensor. ATP binding to isolated T蔚 triggers a major conformational transition. Earlier data were interpreted in terms of an ATP + T蔚_extended 鈫?ATP路T蔚_compact transition that may mimic aspects of the regulatory switching within F₀F₁ (Yagi et al. (2007) Proc. Natl. Acad. Sci. U.S.A., 104, 11233鈥?1238). In this work, we employ complementary biophysical techniques for examining the ATP-induced conformational switching of isolated T蔚. CD spectroscopy confirmed the occurrence of a large-scale conformational transition upon ATP binding, consistent with the formation of stable helical structure. Hydrogen/deuterium exchange (HDX) mass spectrometry revealed that this transition is accompanied by a pronounced stabilization in the vicinity of the ATP-binding pocket. Surprisingly, dramatic stabilization is also seen in the 尾8鈭捨? region, which is remote from the site of ATP interaction. Analytical ultracentrifugation uncovered a previously unrecognized feature of T蔚: a high propensity to undergo dimerization in the presence of ATP. Comparison with existing crystallography data strongly suggests that the unexpected 尾8鈭捨? HDX protection is due to newly formed protein鈥損rotein contacts. Hence, ATP binding to isolated T蔚 proceeds according to 2ATP + 2T蔚_extended 鈫?(ATP路T蔚_compact)₂. Implications of this dimerization propensity for the possible role of T蔚 as an antibiotic target are discussed.