[Show abstract][Hide abstract] ABSTRACT: The membrane protein complex between sarco(endo)plasmic reticulum Ca 2+-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca 2+ affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca 2+ flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults. Heart failure is the leading cause of death worldwide 1. Despite important advances in pharmacological and device therapies, the incidence and the financial impact of this devastating condition is increasing 1. Current drug therapies aim to manage and ameliorate patients' symptoms rather than cure the disease, which has generated a growing emphasis on the development of alternative approaches, including gene therapy 2,3
[Show abstract][Hide abstract] ABSTRACT: Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a "2-color MRP1" construct by fusing GFP and TagRFP to MRP1 nucleotide binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. 2-color MRP1 transport activity was normal, as shown by vesicular transport of [3H]E217βG and doxorubicin efflux in AAV-293 cells. We quantified fluorescence resonance energy transfer (FRET) from GFP to TagRFP as an index of NBD conformational changes. Our results show that ATP binding induces a large-amplitude conformational change that brings the NBDs into closer proximity. FRET was further increased by substrate in the presence of ATP, but not by substrate alone. The data suggest that substrate binding is required to achieve a fully closed and compact structure. ATP analogs bind MRP1 with reduced apparent affinity, inducing a partially closed conformation. The results demonstrate the utility of the 2-color MRP1 construct for investigating ABC transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1.
The American Society for Pharmacology and Experimental Therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Muscle contractility is regulated by a network of many proteins. In cardiomyocytes, the sarco(endo)plasmic reticulum Ca2+ -ATPase, SERCA, and its regulatory protein, phospholamban are responsible for ~70% of Ca2+ reuptake into the SR. While unphosphorylated, PLN inhibits SERCA by lowering its apparent Ca2+ affinity. Upon phosphorylation by PKA at Ser16, PLN inhibition is relieved. This tightly regulated interaction can be easily disrupted by mutation or changes in protein level, leading to heart disease. Thus, understanding the molecular interactions between SERCA/PLN and possible regulators is essential.
Here, we report that ssDNA binds the cytoplasmic domain of PLN with low nanomolar dissociation constants, relieving inhibition of SERCA. The relief of inhibition is length dependent, while affinity is constant for oligonucleotides longer than 10 bases. Solution and solid-state NMR experiments have provided residue specific information that ssDNA targets the cytoplasmic domain of PLN and does not affect SERCA in the absence of PLN. In-cell FRET, and NMR experiments determined that addition of ssDNA does not dissociate PLN from SERCA.
SERCA/PLN has become a highly targeted complex for development of small molecule regulators because of its prevalence in many cardiovascular diseases. While some therapies are currently being investigated, none have proceeded past clinical trials. These results provide a promising avenue for development of novel regulators of the SERCA/PLN complex. Additionally, they support previous findings from our group detailing the intricate balance that is necessary for proper cardiac function.
Biophysical Society National Meeting 2015; 02/2015
[Show abstract][Hide abstract] ABSTRACT: To characterize the conformational dynamics of sarcoplasmic reticulum (SR) calcium pump (SERCA) we performed molecular dynamics simulations beginning with several different high-resolution structures. We quantified differences in structural disorder and dynamics for an open conformation of SERCA versus closed structures, and observed that dynamic motions of SERCA cytoplasmic domains decreased with decreasing domain-domain separation distance. The results are useful for interpretation of recent intramolecular Förster resonance energy transfer (FRET) distance measurements obtained for SERCA fused to fluorescent protein tags. Those previous physical measurements revealed several discrete structural substates and suggested open conformations of SERCA are more dynamic than compact conformations. The present simulations support this hypothesis and provide additional details of SERCA molecular mechanisms. Specifically, all-atoms simulations revealed large-scale translational and rotational motions of the SERCA N-domain relative to the A- and P-domains during the transition from an open to a closed headpiece conformation over the course of a 400 ns trajectory. The open-to-closed structural transition was accompanied by a disorder-to-order transition mediated by an initial interaction of an N-domain loop (Nβ5-β6, residues 426-436) with residues 133-139 of the A-domain. Mutation of three negatively charged N-domain loop residues abolished the disorder-to-order transition, and prevented the initial domain-domain interaction and subsequent closure of the cytoplasmic headpiece. Coarse-grained molecular dynamics simulations were in harmony with all-atoms simulations and physical measurements, and revealed a close communication between fluorescent protein tags and the domain to which they were fused. The data indicate that previous intramolecular FRET distance measurements report SERCA structure changes with high fidelity, and suggest a structural mechanism that facilitates the closure of the SERCA cytoplasmic headpiece.
The Journal of Physical Chemistry B 12/2014; 119(4). DOI:10.1021/jp511433v · 3.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rationale: A naturally occurring missense Leu-39stop (L39X) mutation in phospholamban (PLB) results in truncation of the C-terminal transmembrane domain, leading to cardiomyopathy and premature death in humans.
Objective: The goal of this study was to determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells.
Methods and Results: We fused fluorescent protein tags to PLB and cardiac Ca2+ ATPase (SERCA) to investigate the role of PLB C-terminal residues for membrane localization, PLB oligomerization and SERCA regulation. Alanine substitution of C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB. Notably, substitution mutation V49A had profound effects on pentamer structure and regulatory complex conformation, increasing and decreasing probe separation distance, respectively. Progressive deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Molecular dynamics simulations recapitulated decreased bilayer anchoring for truncated PLB. C-terminal truncations resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCA-PLB binding affinity, and loss of inhibitory potency as quantified by Ca2+-dependent ATPase activity. However, despite decreased SERCA-PLB binding, intermolecular FRET was paradoxically increased as a result of a 14.5 Å decrease in the distance between donor and acceptor fluorophores.
Conclusions: We conclude that PLB C-terminal residues are critical for membrane anchoring and quaternary structure determination of PLB pentamer and PLB-SERCA regulatory complex. The loss of membrane registration restraint by C-terminal residues (especially V49) causes displacement of PLB to an alternative position on SERCA. The data are compatible with a model in which PLB binds to the canonical inhibitory binding site and an additional novel site.
[Show abstract][Hide abstract] ABSTRACT: Rationale: A naturally-occurring, missense Arg9-to-Cys (R9C) mutation of phospholamban (PLB) triggers cardiomyopathy and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of R9C-PLB in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) regulation and cardiomyocyte Ca2+ handling is not clear.
Objective: The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte Ca2+ kinetics and contractility and identify the molecular mechanism underlying R9C pathology.
Methods and Results: We measured the physiological consequences of R9C-PLB mutation on Ca2+ transients and sarcomere shortening in adult cardiomyocytes at increasing pacing frequencies. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we fused fluorescent protein tags to PLB and SERCA, and compared the effect of R9C and pentamer-destabilizing mutation (SSS) on PLB oligomerization and PLB-SERCA interaction. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C exhibited an increased affinity of PLB oligomerization, and a decreased binding affinity to SERCA due to an oxidative modification which mimics phosphorylation. Real-time FRET analysis in cardiomyocytes revealed that R9C-PLB exhibits enhanced sensitivity to oxidative stress, which is a prevailing condition in heart failure.
Conclusions: We conclude that the primary mechanism of R9C pathology is a phosphomimetic effect of PLB cysteine oxidation, manifested as increased oligomerization and a change in the structure of the PLB-SERCA regulatory complex.
[Show abstract][Hide abstract] ABSTRACT: TRIM5 alpha proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5 alpha proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5 alpha molecules within these assemblies have remained unclear. Here we show that alpha-helical elements within the Linker2 region of rhesus macaque TRIM5 alpha govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce alpha-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the alpha-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5 alpha dimer suggests a model in which alpha-helical elements within the Linker2 region dock to alpha-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5 alpha assembly and restriction and demonstrate that the antiviral potency of TRIM5 alpha proteins can be significantly increased without altering the affinity of SPRY/capsid binding.
Journal of Virology 08/2014; 88(16):8911-23. DOI:10.1128/JVI.01134-14 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium A TPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. V al to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB- SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET, due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA, and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for V al49-stop (V49X) truncation mutant was paradoxically increased as a result of a 11.3 angstrom decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C-terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.
[Show abstract][Hide abstract] ABSTRACT: We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.
[Show abstract][Hide abstract] ABSTRACT: On a cellular level, the cardiac cycle is orchestrated by release of Ca2+ into the cytosol during systole (contraction) and subsequent reuptake of Ca2+ into internal stores by an ion-motive ATPase during diastole (relaxation). This pump, the sarco(endo)plasmic reticulum calcium ATPase (SERCA), undergoes large conformational changes between enzymatic substates during calcium transport. Recently, we quantified SERCA structural dynamics using intramolecular fluorescence resonance energy transfer (FRET). Here, we extend this work to investigate SERCA conformational changes in atomic detail using molecular dynamics simulations. Specifically, we tested a hypothesis generated from FRET experiments: Open conformations of SERCA are more dynamic than compact, closed structures. Our molecular dynamics simulations showed that larger amplitude cytoplasmic domain motions for open conformations compare to compact structures. The data suggest that the open conformation of SERCA is more dynamically disordered than the closed state. We also performed a molecular dynamics simulation on PLB, a homopentameric regulator of SERCA. Previous studies have suggested that a naturally occurring human heart failure mutation of PLB, L39Stop, disrupts membrane localization. Molecular dynamics simulations showed that L39Stop-PLB was unstable compared to WT PLB. In particular, we observed that the interactions between protomers of the L39Stop PLB pentamer were disrupted within 10 ns, resulting in collapse of the pentamer structure. L39Stop-PLB monomers were subsequently extruded from the bilayer, becoming soluble in the aqueous phase. We determined that the instability of the L39Stop PLB pentamers is partially due to lack of van der Waals interactions between truncated PLB monomers.
[Show abstract][Hide abstract] ABSTRACT: A naturally occurring missense Leu39stop (L39X) mutation in phospholamban (PLB) results in truncation of the Cterminal transmembrane domain, leading to cardiomyopathy and premature death. In this study, we fused PLB and SERCA to fluorescent protein tags to determine the structural and thermodynamic consequences of progressive truncations of the Cterminal residues of PLB in the membranes of living cells. We found that deletion of only a few Cterminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. Selective permeabilization of the plasma membrane by saponin resulted in diffusion of fluorescently labeled PLB out of the cells, consistent with solubilization of truncated proteins. Western blot analysis showed the expected mobilities for truncation mutants relative to full length PLBWT, indicating that the observed solubilization of PLB truncation mutants is not due to proteolysis. Moreover, molecular dynamics simulations recapitulated the observed loss of stable bilayer anchoring for truncated PLB. Fluorescence resonance energy transfer (FRET) analysis revealed that Cterminal truncations resulted in progressive loss of PLBPLB FRET, indicating a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the SERCAPLB binding affinity. Despite this decrease in affinity, SERCAPLB FRET was paradoxically increased by deletion of up to 4 Cterminal residues as a result of a 14 angstrom decrease in the distance between donor and acceptor fluorophores. Truncation of PLB also decreased its inhibitory potency as quantified by calciumdependent ATPase activity. We conclude that the Cterminal residues are critical for PLB localization, SERCAPLB regulatory complex conformation, and PLB regulatory function.
[Show abstract][Hide abstract] ABSTRACT: The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca2+ handling and contractility by reversibly inhibiting SR Ca2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization.
Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation.
Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization.
Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.
Circulation Research 08/2013; 113(4). · 11.09 Impact Factor