What Can We Learn from a Small Regulatory Membrane Protein?
This chapter reviews the molecular biology, biochemical, and NMR methods that we used to study the structural dynamics, membrane topology, and interaction of phospholamban (PLN), a small regulatory membrane protein involved in the regulation of the sarcop
- PDF / 2,041,274 Bytes
- 17 Pages / 504 x 720 pts Page_size
- 14 Downloads / 200 Views
1. Introduction Phospholamban (PLN) is a small integral membrane protein (52 residues) localized in the sarcoplasmic reticulum (SR) membrane that regulates the flow of calcium ions into the SR lumen of cardiac muscle (1). Specifically, PLN binds to SR calcium ATPase (SERCA), the enzyme responsible for calcium re-uptake into the SR lumen. In its unphosphorylated form, PLN inhibits SERCA by reducing its affinity for calcium. Upon b-adrenergic stimulation, PLN is phosphorylated by protein kinase A at S16 with concomitant relief of SERCA inhibition and restoration of SERCA activity. These cyclic events account for the relaxation (diastolic phase) of the heart muscle, which if disrupted, may evolve into heart Jean-Jacques Lacapère (ed.), Membrane Protein Structure Determination: Methods and Protocols, Methods in Molecular Biology, vol. 654, DOI 10.1007/978-1-60761-762-4_16, © Springer Science+Business Media, LLC 2010
303
304
Veglia et al.
failure (2). Due to their central role in cardiac muscle contractility, PLN and SERCA have become targets for new, alternative therapies for heart failure (3). What can be learned from the study of the structural and dynamic transitions of PLN free and bound to SERCA?, More importantly, can these studies contribute in the development of new clinical approaches to heart failure? Much progress has been made toward understanding the molecular mechanism of SERCA within the enzymatic cycle (4, 5). Chapters 9 and 15 of this volume give exquisite portraits of the latest findings, outlining the successes of x-ray crystallography in reconstructing the structural transitions of SERCA during enzyme turnover. Undeniably, the first crystal structure of SERCA (E1-Ca2, calcium-bound form) solved by Toyoshima and co-workers paved the way for the atomic level understanding of calcium translocation in the SR (6). To date, there are several crystal structures of SERCA under different experimental conditions that are allowing enzymologists to model the major conformational states of the enzyme during turnover (7). However, very few studies have been dedicated to the determination of the complexes between SERCA and its endogenous inhibitor PLN. This is probably due to the inherent dynamics of the SERCA/PLN complex that might complicate the formation of large, diffracting crystals. This hypothesis is reinforced by several fluorescence and EPR studies published by the Thomas and Squier Laboratories underscoring the dynamic nature of the SERCA/PLN complex (8–14). So far, the labs of Stokes and Young (15, 16) have produced the best images of this complex although the quality of the crystals does not define the interaction surface between the two proteins at the atomic level. NMR studies, on the other hand, have been focused on the structural dynamics of PLN in the unbound form. This is mainly due to the difficulties in producing isotopically labeled SERCA and analyzing its complex with PLN by solution-state NMR (Mw ~ 116 kDa). Our laboratory, in collaboration with the Thomas laboratory, has used a combinati
Data Loading...
