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Interactions between Ca²⁺-ATPase and the Pentameric Form of Phospholamban in Two-Dimensional Co-Crystals

David L. Stokes ^* , Andrew J. Pomfret ^*, William J. Rice , John Paul Glaves  and Howard S. Young 

^* Structural Biology Program, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York 10016; New York Structural Biology Center, New York, New York 10027; and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

Correspondence: Address reprint requests to Howard S. Young, Tel.: 780-492-3931; Fax: 780-492-0095; E-mail: hyoung{at}ualberta.ca.

Phospholamban (PLB) physically interacts with Ca²⁺-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca²⁺-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca²⁺-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22₁2₁ symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca²⁺-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca²⁺-ATPase.

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