title: Tuning the structural coupling between the transmembrane and cytoplasmic domains of phospholamban to control sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) function.
abstract: Phospholamban (PLN) is the endogenous inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), the integral membrane enzyme responsible for 70 % of the removal of Ca(2+) from the cytosol, inducing cardiac muscle relaxation in humans. Dysfunctions in SERCA:PLN interactions have been implicated as having a critical role in cardiac disease, and targeting Ca(2+) transport has been demonstrated to be a promising avenue in treating conditions of heart failure. Here, we designed a series of new mutants able to tune SERCA function, targeting the loop sequence that connects the transmembrane and cytoplasmic helices of PLN. We found that a variable degree of loss of inhibition mutants is attainable by engineering glycine mutations along PLN's loop domain. Remarkably, a double glycine mutation results in a complete loss-of-function mutant, fully mimicking the phosphorylated state of PLN. Using nuclear magnetic resonance spectroscopy, we rationalized the effects of these mutations in terms of entropic control on PLN function, whose inhibitory function can be modulated by increasing its conformational dynamics. However, if PLN mutations go past a threshold set by the phosphorylated state, they break the structural coupling between the transmembrane and cytoplasmic domains, resulting in a species that behaves as the inhibitory transmembrane domain alone. These studies provide new potential candidates for gene therapy to reverse the effects of heart failure.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Kim N
authors.initials: KN
authors.lastname: Ha
authors.forename: Martin
authors.initials: M
authors.lastname: Gustavsson
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Veglia
mesh_terms: D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D005998:Glycine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban