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ATP-induced dimerization of the F0F1 ε subunit from Bacillus PS3: a hydrogen exchange-mass spectrometry study.

Abstract：

: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 ε subunit is involved in regulating this ATPase activity. Also, ε is essential for coupling ATP hydrolysis (or synthesis) to proton translocation. The ε subunit consists of a β sandwich and two C-terminal helices, α1 and α2. The protein can switch from a compact fold to an alternate conformation where α1 and α2 are separated, resulting in an extended structure. ε from the thermophile Bacillus PS3 (Tε) binds ATP with high affinity such that this protein may function as an intracellular ATP level sensor. ATP binding to isolated Tε triggers a major conformational transition. Earlier data were interpreted in terms of an ATP + Tεextended → ATP·Tεcompact transition that may mimic aspects of the regulatory switching within F0F1 (Yagi et al. (2007) Proc. Natl. Acad. Sci. U.S.A., 104, 11233–11238). In this work, we employ complementary biophysical techniques for examining the ATP-induced conformational switching of isolated Tε. 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 β8−β9 region, which is remote from the site of ATP interaction. Analytical ultracentrifugation uncovered a previously unrecognized feature of Tε: a high propensity to undergo dimerization in the presence of ATP. Comparison with existing crystallography data strongly suggests that the unexpected β8−β9 HDX protection is due to newly formed protein–protein contacts. Hence, ATP binding to isolated Tε proceeds according to 2ATP + 2Tεextended → (ATP·Tεcompact)2. Implications of this dimerization propensity for the possible role of Tε as an antibiotic target are discussed.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Rodriguez AD,Dunn SD,Konermann L

doi

10.1021/bi5004684

subject

Has Abstract

pub_date

2014-06-24 00:00:00

pages

4072-80

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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ATP-induced dimerization of the F0F1 ε subunit from Bacillus PS3: a hydrogen exchange-mass spectrometry study.