Single-Molecule Dynamics Reveal an Altered Conformation for the Autoinhibitory Domain of Plasma Membrane Ca2+-ATPase Bound to Oxidatively Modified Calmodulin

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• 作者：Kenneth D. Osborn ; Ryan K. Bartlett ; Abhijit Mandal ; Asma Zaidi ; Ramona J. Bieber Urbauer ; Jeffrey L. Urbauer ; Nadya Galeva ; Todd D. Williams ; and Carey K. Johnson
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：October 12, 2004
• 年：2004
• 卷：43
• 期：40
• 页码：12937 - 12944
• 全文大小：95K
• 年卷期：v.43,no.40(October 12, 2004)
• ISSN：1520-4995

文摘

We used single-molecule polarization modulation methods to investigate the activation of theplasma membrane Ca²⁺-ATPase (PMCA) by oxidized calmodulin (CaM). Oxidative modification ofmethionine residues of CaM to their corresponding sulfoxides is known to inhibit the ability of CaM toactivate PMCA. Single-molecule polarization methods were used to measure the orientational mobilityof fluorescently labeled oxidized CaM bound to PMCA. We previously identified two distinct populationsof PMCA-CaM complexes characterized by high and low orientational mobilities, with the low-mobilitypopulation appearing at a subsaturating Ca²⁺ concentration [Osborn, K. D., et al. (2004) Biophys. J. 87,1892-1899]. We proposed that the high-mobility population corresponds to PMCA-CaM complexeswith a dissociated (and mobile) autoinhibitory domain, whereas the low-mobility population correspondsto PMCA-CaM complexes where the autoinhibitory domain is not dissociated and therefore the enzymeis not active. In the present experiments, performed with PMCA complexed with oxidatively modifiedCaM at a saturating Ca²⁺ concentration, we found a large population of molecules with an orientationallyimmobile autoinhibitory domain. In contrast, native CaM bound to PMCA was characterized almost entirelyby the more orientationally mobile population at a similar Ca²⁺ concentration. The addition of 1 mMATP to complexes of oxidized CaM with PMCA reduced but did not abolish the low-mobility population.These results indicate that the decline in the ability of oxidized CaM to activate PMCA results at least inpart from its reduced ability to induce conformational changes in PMCA that result in dissociation of theautoinhibitory domain after CaM binding.