The overall goal of the proposal is to understand calcium regulation in both cardiac and skeletal muscle at the atomic level. Calcium is an essential messenger for muscle contractility and its homeostatic balance is controlled by proteins embedded in the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca2+-ATPase 2a isoform (SERCA2a) is responsible for ~70% of the Ca2+ translocation and regulates diastole. SERCA2a is inhibited by phospholamban (PLN), a membrane protein that reverses its inhibition upon phosphorylation at Ser16 and Thr17. In skeletal muscle, the SERCA1a isoform administrates the relaxation phase and is regulated by sarcolipin (SLN), a membrane inhibitor that is post-translationally regulated by phosphorylation at Thr5. Both PLN and SLN maintain SERCA's activity within a physiological window of apparent Ca2+ affinity. When SERCA2a functions outside this window, disruptions in Ca2+ homeostasis leads to dilated or hypertrophic cardiomyopathies, and ultimately heart failure. SERCA1a dysfunctions result in reduced skeletal contractility, leading to conditions such as Brody disease. In past funding cycles, we characterized the structural dynamics of both PLN and SLN in the presence and absence of the ATPase. The latter enabled us to design and test new dominant-negative PLN mutants with promising results towards improving muscle contractility via rAAV-mediated gene therapy. In this competitive renewal, we propose to analyze the effects of the phosphorylation states in both PLN and SLN through investigating the interactions between these two inhibitors and the enzyme along the catalytic cycle, and take advantage of this knowledge in designing mutants with improved loss-of-function characteristics. To carry out these studies, we will utilize a combination of molecular biology, biochemical assay, as well as spectroscopic methods (NMR, EPR, and fluorescence) that will enable the analysis of these membrane protein complexes in native lipids.

Public Health Relevance

The proposal focuses on the structural analysis of the interactions between the sarcoplasmic reticulum Ca2+-ATPase and its two inhibitors, phospholamban and sarcolipin. Correct functioning of these of these protein complexes is directly linked with cardiac and skeletal muscle diseases, such as dilated cardiomyopathy, hypertrophy, and Brody disease. Understanding the structural details of these interactions will be instrumental to designing innovative therapies to treat these devastating diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064742-11
Application #
8517134
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Wehrle, Janna P
Project Start
2002-09-16
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
11
Fiscal Year
2013
Total Cost
$435,843
Indirect Cost
$144,301
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
De Simone, Alfonso; Mote, Kaustubh R; Veglia, Gianluigi (2014) Structural dynamics and conformational equilibria of SERCA regulatory proteins in membranes by solid-state NMR restrained simulations. Biophys J 106:2566-76
Vostrikov, Vitaly V; Mote, Kaustubh R; Verardi, Raffaello et al. (2013) Structural dynamics and topology of phosphorylated phospholamban homopentamer reveal its role in the regulation of calcium transport. Structure 21:2119-30
De Simone, Alfonso; Gustavsson, Martin; Montalvao, Rinaldo W et al. (2013) Structures of the excited states of phospholamban and shifts in their populations upon phosphorylation. Biochemistry 52:6684-94
Mote, Kaustubh R; Gopinath, T; Veglia, Gianluigi (2013) Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy. J Biomol NMR 57:91-102
Gustavsson, Martin; Verardi, Raffaello; Mullen, Daniel G et al. (2013) Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban. Proc Natl Acad Sci U S A 110:17338-43
Gopinath, T; Mote, Kaustubh R; Veglia, Gianluigi (2013) Sensitivity and resolution enhancement of oriented solid-state NMR: application to membrane proteins. Prog Nucl Magn Reson Spectrosc 75:50-68
Gopinath, T; Veglia, Gianluigi (2013) Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra. J Chem Phys 138:184201
Porcelli, Fernando; Ramamoorthy, Ayyalusamy; Barany, George et al. (2013) On the role of NMR spectroscopy for characterization of antimicrobial peptides. Methods Mol Biol 1063:159-80
Gustavsson, Martin; Traaseth, Nathaniel J; Veglia, Gianluigi (2012) Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy. Biochim Biophys Acta 1818:146-53
Gopinath, T; Veglia, Gianluigi (2012) Dual acquisition magic-angle spinning solid-state NMR-spectroscopy: simultaneous acquisition of multidimensional spectra of biomacromolecules. Angew Chem Int Ed Engl 51:2731-5

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