Cornea, R. L. | Search bioRxiv

bioRxiv2020

A Bitopic Miniprotein Regulates a Membrane-Embedded Enzyme via Topological Allostery

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to {beta}-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzymes function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLNs cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzymes active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

biophysics↗

bioRxiv2019

SERCA2a-activating PLB mutant improves calcium handling in dilated cardiomyopathy model of hiPSC-CMs

There is increasing momentum toward the development of gene therapy for heart failure (HF), cardiomyopathy, and other progressive cardiac diseases that correlate with impaired calcium (Ca2+) transport and reduced contractility. We have used FRET between fluorescently-tagged SERCA2a (the cardiac Ca2+ pump) and PLB (its ventricular peptide inhibitor) to test directly the effectiveness of loss-of-inhibition/gain-of-binding (LOI/GOB) PLB mutants (PLBM) that were engineered to compete with the binding of inhibitory wild type PLB (PLBWT). Our therapeutic strategy is to relieve PLBWT inhibition of SERCA2a by utilizing the reserve adrenergic capacity of PLB to enhance baseline cardiac contractility. Using a FRET assay, we determined that the combination of a LOI PLB mutation (L31A) and a GOB PLB mutation (I40A) results in a novel engineered LOI/GOB PLBM (L31A/I40A) that effectively competes with PLBWT binding to cardiac SERCA2a in HEK293-6E cells. We demonstrated that co-expression of L31A/I40A-PLBM enhances SERCA Ca-ATPase activity by increasing enzyme Ca2+ affinity (1/KCa) in PLBWT-inhibited HEK cell homogenates. For an initial assessment of PLBM physiological effectiveness, we used human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) from a healthy individual. In this system, we observed that adeno-associated virus 2 (rAAV2)-driven expression of L31A/I40A-PLBM enhances the amplitude of SR Ca2+ release and the rate of SR Ca2+ re-uptake. To assess therapeutic potential, we used an hiPSC-CM model of dilated cardiomyopathy (DCM) containing PLB mutation R14del, where we observed that rAAV2-driven expression of L31A/I40A-PLBM rescues arrhythmic Ca2+ transients and alleviates decreased Ca2+ transport. Based on these results, PLBM transgene expression is a promising gene therapy strategy for cardiomyopathies associated with impaired Ca2+ transport and decreased contractility.

biophysics↗