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Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach

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Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach

Content Provider	Springer Nature Link
Author	Mayor, June A. Sun, Jiakang Kotaria, Rusudan Walters, D. Eric Oh, Kyoung Joon Kaplan, Ronald S.
Copyright Year	2010
Abstract	The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter’s two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5′-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.
Starting Page	99
Ending Page	109
Page Count	11
File Format	PDF
ISSN	0145479X
Journal	Journal of Bioenergetics and Biomembranes
Volume Number	42
Issue Number	2
e-ISSN	15736881
Language	English
Publisher	Springer US
Publisher Date	2010-03-31
Publisher Place	Boston
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Citrate transporter Liposomes Mitochondria Membrane proteins Site-directed spin labeling Electron paramagnetic resonance Inhibitors Organic Chemistry Animal Biochemistry Animal Anatomy / Morphology / Histology Biochemistry Bioorganic Chemistry
Content Type	Text
Resource Type	Article
Subject	Cell Biology Physiology

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