Spectroscopic Investigation of Phosphorylated Pentameric Phospholamban Bound to the Sarcoplasmic Calcium ATPase

Abstract

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) is the main enzyme responsible for the reuptake of Ca2+ into the sarcoplasmic reticulum (SR) during muscle relaxation. Phospholamban (PLN), a homopentameric membrane protein expressed in the SR, binds to and decreases the rate of Ca2+ transport of SERCA. This inhibitory action is relieved when PLN is phosphorylated at residues Ser-16 or Thr-17 in response to different signal pathways. Although a great deal of knowledge has been accumulated on the functional consequences of PLN phosphorylation, very little is known about the molecular details leading to relief of inhibition. Previous work suggested a crucial role of protein topology in the complex between SERCA and PLN, with the latter showing different tilt angles in the presence and absence of SERCA. Our hypothesis is that SERCA activity is regulated by changes in topology in PLN transmembrane helices upon phosphorylation. In order to probe for the orientation of protein domains, solid-state NMR in oriented lipid bilayers has established itself as the method of choice in many studies. Here we present data on the topological changes taking place in phospholamban pentamer in the presence of SERCA upon phosphorylation at Ser-16 or Thr-17. SERCA and PLN are reconstituted in lipid bilayers and uniaxially aligned either mechanically (on glass plates) or magnetically (upon formation of bicelles). The two proteins are fully functional under these conditions as demonstrated by the ability of SERCA to transport Ca2+ in functional assays as well as PLN dependent decrease in apparent affinity for Ca2+.