PNNL

Abstract

Mutagenesis and cross-linking measurements have identified specific contact interations between the cytosolic and transmembrane sequences of phospholamban (PLB) and the Ca-ATPase, and, in conjunction with the high resolution structures of PLB and the Ca-ATPase, have been used to construct models of the PLB-ATPase complex, which suggest PLB adopts a more extended structure within this complex. To directly test these predictions, we have used fluorescence resonance energy transfer to measure the average conformation and heterogeneity between chromophores covalently bound to the transmembrane and cytosolic domain of PLB assumes a wide range of structures relative to the transmembrane sequence, which can be described using a model involving a Gaussian distribution of distances with an average distance (Rav)less than 21A and a half-width (HW) of 36A. This conformational heterogeneity of PLB is consistent with the ten structures resolved by NMR for the C41F mutant of PLB in organic co-solvents. In contrast, PLB bound to the Ca-ATPase assumes a unique and highly ordered confirmation, where Rav-14.0+0l.3A and HW=3.7+0.6A. The small spatial separation between the bound chromophores on PLB is inconsistent with an extended conformation of bound PLB in current models. Thus, to satisfy known interaction sites of PLB and the Ca-ATPase, these findings suggest a re-orientation of the nucleotide binding domain of the Ca-ATPase toward the bilayer surface in order to bring known PLB binding sites into close juxtaposition with residues near the amino-terminus of PLB. Induction of an altered conformation of the nucleotide binding domain of the Ca-ATPase by PLB binding is suggested to underly the reduced calcium sensitivity associated with PLB inhibition of the pump.