The long term goal ofthis research is to elucidate the molecular mechanism by which phospholamban (PLB) inhibits the Capump (SERCAaa isoform) in cardiac sarcoplasmic reticulum (SR). PLBis a pentameric phosphoprotein of five identical monomers in cardiac SR. The PLBmonomer is responsible for binding to SERCAaa and inhibiting it. Wepropose to localize the binding-interaction sites between the PLB monomer and SERCA2a that lead to enzymeinhibition, and to determine howthis protein interaction is regulated by key allosteric modulators including Ca concentration, nucleotides, and PLBphosphorylation. Emphasis will be placed upon identifying aminoacids within the inhibited complex that interact directly,taking advantage of our newlydeveloped chemical cross-linking method.
In Aim i, we will perform Cys-scanning mutagenesis of PLBto localize distinct sites along its primary structure that cross-link to endogenousCys residues of SERCA2a.
In Aim 2, Lysresidues of SERCA2a that cross-link to distinct sites of PLBwill be localized. Using cross-linking agents as molecular rulers, we willthen develop an accurate 3-D model of the quaternary structure ofthe PLBmonomer bound to SERCA2a.
In Aim 3, the effects of Caconcentration, nucleotides, and the inhibitor thapsigargin on cross-linking of PLBto SERCAaa will be investigated. The hypothesis tested is that PLB binds exclusivelyto the Ca-free form (?2) of SERCA2a, but only?2 stabilized by nucleotides CE2-ATP).
In Aim 4, we will determine how Ca reverses PLBinhibition of SERCA2a. We propose that PLBand Cabinding to SERCA2a are mutually exclusive. Cabinding to El favors global dissociation of PLBfrom .E2-ATP, thus allowingthe Capump to be freely active and transport Ca at the maximal rate. The Ca-binding site of SERCAaa responsible for dissociating PLBwillbe identified, and the effect of PLB on the Ca-bindingaffinity of SERCA2a willbe quantified.
In Aim 5, we will determine how phosphorylation of PLB by protein kinases reverses PLBinhibition. The hypothesis tested is that phosphorylation of PLBby protein kinases causes it to fully dissociate from SERCA2a at low ionizedCa concentration, thus allowing Catransport to proceed unencumbered. PLBis a key regulator of cardiac contractility. Bydefining its molecular mechanism of action, new insights on PLBregulation of the strength of the heartbeat will result, whichmay ultimately lead to the design of new drugs to treat heart failure.
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|Akin, Brandy L; Chen, Zhenhui; Jones, Larry R (2010) Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state. J Biol Chem 285:28540-52|
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