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J. Biol. Chem., Vol. 282, Issue 51, 37205-37214, December 21, 2007

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Controlling the Inhibition of the Sarcoplasmic Ca²⁺-ATPase by Tuning Phospholamban Structural Dynamics^*

Kim N. Ha, Nathaniel J. Traaseth¹, Raffaello Verardi, Jamillah Zamoon, Alessandro Cembran, Christine B. Karim, David D. Thomas, and Gianluigi Veglia²

From the Department of Chemistry and Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca²⁺-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.

Received for publication, May 16, 2007 , and in revised form, September 26, 2007.

^* This work was supported in part by the National Institutes of Health Grants GM64742 and K02HL080081 (to G. V.) and GM27906 (to D. D. T.). NMR instrumentation at the University of Minnesota High Field NMR Center was funded by National Science Foundation Grant BIR-961477 and the University of Minnesota Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by American Heart Association Greater Midwest Affiliate predoctoral fellowship 0515491Z.

² To whom correspondence should be addressed: Dept. of Biochemistry, Biophysics, and Molecular Biology, University of Minnesota, 6-155 Jackson Hall, Minneapolis, MN 55455. Tel.: 612-625-0758; Fax: 612-625-2163; E-mail: veglia{at}chem.umn.edu.

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				E. M. Kelly, Z. Hou, J. Bossuyt, D. M. Bers, and S. L. Robia Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells J. Biol. Chem., May 2, 2008; 283(18): 12202 - 12211. [Abstract] [Full Text] [PDF]