Controlling the inhibition of the sarcoplasmic Ca2+-ATPase by tuning phospholamban structural dynamics.
Cardiac contraction and relaxation are regulated by conformational transitions of protein complexes that are responsible for calcium trafficking through cell membranes. Central to the muscle relaxation phase is a dynamic membrane protein complex formed by Ca2+-ATPase (SERCA) and phospholamban (PLN), which in humans is responsible for approximately 70% of the calcium re-uptake in the sarcoplasmic reticulum. Dysfunction in this regulatory mechanism causes severe pathophysiologies. In this report, we used a combination of nuclear magnetic resonance, electron paramagnetic resonance, and coupled enzyme assays to investigate how single mutations at position 21 of PLN affects its structural dynamics and, in turn, its interaction with SERCA. We found that it is possible to control the activity of SERCA by tuning PLN structural dynamics. Both increased rigidity and mobility of the PLN backbone cause a reduction of SERCA inhibition, affecting calcium transport. Although the more rigid, loss-of-function (LOF) mutants have lower binding affinities for SERCA, the more dynamic LOF mutants have binding affinities similar to that of PLN. Here, we demonstrate that it is possible to harness this knowledge to design new LOF mutants with activity similar to S16E (a mutant already used in gene therapy) for possible application in recombinant gene therapy. As proof of concept, we show a new mutant of PLN, P21G, with improved LOF characteristics in vitro.
Ha
Kim N
KN
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Traaseth
Nathaniel J
NJ
Verardi
Raffaello
R
Zamoon
Jamillah
J
Cembran
Alessandro
A
Karim
Christine B
CB
Thomas
David D
DD
Veglia
Gianluigi
G
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D015316:Genetic Therapy; D006331:Heart Diseases / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology / Q000628:therapy; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009200:Myocardial Contraction / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D046912:Multiprotein Complexes; D009124:Muscle Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban