In adult cardiomyocytes, the type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a vital role in intracellular Ca2+ regulation. Reduced SERCA2a function has been associated with decreased myocardial contraction and cardiac output in several heart diseases. Consequently, increasing SERCA2a activity is a high-priority target for treating cardiac pathologies associated with abnormal Ca2+ homeostasis. In our previous SERCA ATPase-based screening study, we identified several small molecules as potential activators of SERCA2a function, including Compound 9, a piperidinyl amide. With porcine cardiac sarcoplasmic reticulum (SR) preparations, we confirmed activation of both SERCA2a ATPase and Ca2+-uptake activities. In the current study, we analyzed the effect of Compound 9 on SERCA2a activity on intracellular Ca2+ dynamics in ventricular myocytes. Using FRET with human SERCA2a overexpressed in mammalian cells, we confirm that Compound 9 binds and alters SERCA structural dynamics independent of peptide regulators, including phospholamban (PLB). Confocal microscopy and in-cell Ca2+ imaging revealed that Compound 9 enhanced Ca2+ dynamics in mouse ventricular myocytes. Compound 9 (10 μM) increased the action potential-induced Ca2+ transients by 65% and SR Ca2+ load by 29%. Moreover, Compound 9 increased Ca2+ dynamics during adrenergic receptor stimulation and in PLB knockout cardiomyocytes, suggesting the stimulatory effect of Compound 9 is PLB independent. Overall, Compound 9 displays characteristics that can be beneficial to enhance cardiac intracellular Ca2+ dynamics by increasing SERCA2a function.

We have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activity in vitro , yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the same a - and d -arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on the e -arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that the e -arm and hydrophobic residues in the adjacent b -arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN‒SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a central regulator of cardiac Ca2+ handling and an emerging therapeutic target for heart failure. Here, we report a comprehensive structure-activity relationship (SAR) study around small-molecule activator compound 1, integrating Ca2+-ATPase and Ca2+-uptake assays, isoform selectivity profiling, and ADMET characterization across more than fifty analogues. Systematic modification of the left-hand aryl/heteroaryl region revealed a strong dependence of activity on aromaticity and lipophilicity, with CF3- and Br-substituted analogues providing substantial gains in potency. Optimization of the central amide linker established the importance of N-alkyl chain length, subtle hydrogen-bonding capacity, and a bent ligand geometry for productive SERCA2a engagement. Electronic tuning of the right-hand benzyl group further modulated efficacy, highlighting the essential contribution of an ortho-donor substituent. Functional evaluation across multiple Ca2+ concentrations identified several analogues with ATPase activation but inhibitory Ca2+-uptake effects, underscoring the need for dual-assay assessment to ensure bona fide activation. Among the series, compound 25 emerged as a balanced lead, displaying micromolar potency, robust concordant enhancement of ATPase and Ca2+-uptake activity, favorable solubility, and improved cytotoxicity relative to compound 1. Collectively, these findings define key structural determinants governing SERCA2a activation and provide a rational framework for developing next-generation, drug-like cardiac SERCA2a modulators.

Myosin disordered‐ and super‐relaxed states (DRX and SRX, respectively) in skeletal muscle fibers are hypothesized to play key roles in thermogenesis and basal metabolic energy expenditure, raising potential for novel therapeutic targets for obesity and other metabolic diseases. Limited studies have investigated relationships between body composition or biological sex and myosin relaxed states. Using fluorescence‐based single‐nucleotide turnover, we report quantitative relationships of diet‐induced adiposity and sex with biochemical parameters of myosin relaxed states of rodent muscle fibers. Our main findings were: (1) adiposity had minimal to no effect on parameters of relaxed myosin states measured in fibers from rats and mice, (2) fibers from female rats and mice had 10%–20% shorter SRX lifetimes than those from males ( p ≤ 0.035), (3) in rats, females had shorter DRX lifetimes than males, and (4) myosin heavy chain isoform had negligible impact on parameters of relaxed myosin states. We conclude that skeletal muscle energy utilization during rest, as measured by myosin ATPase, is affected minimally by adiposity, but differs by sex. Continued exploration of the metabolic implications of myosin transitioning between SRX and DRX will provide further understanding of muscle thermogenesis and whole‐body metabolism; in so doing, sex as a biological factor should be considered.

The oncoprotein c-Myc is overexpressed or mutated in a large fraction of human cancers. The stability of c-Myc is controlled by phosphorylation of T58 and S62 within a conserved degron motif in the N-terminal transactivation domain, which triggers recruitment of the SCF ubiquitin ligase. The kinase Aurora A (AurA) has been shown to bind to both c-Myc and its paralog N-Myc and to promote their stability by interfering with ubiquitination and degradation. Here we show, using NMR and FRET experiments, that AurA binds to c-Myc through several discrete interactions spanning 145 residues within its transactivation domain. AurA binding to c-Myc is enhanced by phosphorylation of the T58/S62 degron, demonstrating that the kinase recognizes the pool of c-Myc that has been marked for degradation by the ubiquitin proteasome pathway. Although AurA binds to segments of c-Myc flanking the degron, it does not appear to form extensive interactions with the phosphorylated degron itself, potentially leaving it accessible on the AurA surface. These observations establish a foundation for understanding the role of AurA in regulating c-Myc ubiquitination and degradation.

We are engineering cell-based high-throughput screening (HTS) methods using time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and structural dynamics of the SERCA2a/DWORF complex. Previous work from our group has focused on the membrane protein complex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its inhibitor phospholamban (PLB). This complex is a validated therapeutic target for reversing contractile dysfunction caused by aberrant calcium handling in the heart. We are currently employing an analogous approach to identify drug molecules that promote the binding of the activator dwarf open reading frame (DWORF) to SERCA2a. DNA constructs have been made to either co-express mMaroon-SERCA2a and mCyRFP-DWORF or express them as a fusion protein complex in the endoplasmic reticulum of HEK293 cells. Using a unique fluorescence lifetime microplate reader (FLT-PR), which increases the throughput of high-precision FLT measurements by several orders of magnitude, we can measure FRET between mMaroon-SERCA2a (donor) and mCyRFP-DWORF (acceptor). A triplicate screen against the 2700 SELLECK library of FDA-approved compounds will allow for identification of hit compounds that reproducibly change FRET. In previous studies of the SERCA2a/PLB complex, we sought compounds that decreased FRET, as this implied a (partial) dissociation of PLB and subsequent increase in SERCA2a calcium affinity. In contrast, for the SERCA2a/DWORF complex, an increase in FRET may correlate with improved binding of DWORF and an increase in SERCA2a activity. Hit compounds will be subjected to SERCA2a functional assays to determine if the observed increase in FRET does indeed correlate with an increase in Ca-ATPase activity, potentially leading to identification of a drug that could reverse calcium mishandling, as required for treatment of heart disease. This work is supported by grants NIH R01HL139065 (to DDT/RTR) and T32AR007612 (to DDT).

Calmodulin (CaM) activates the skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1) at nanomolar Ca2+ and inhibits it at micromolar Ca2+. CaM conversion from RyR1 activator to inhibitor is due to structural changes induced by Ca2+ binding at CaM's two lobes. However, it remains unclear which lobe provides the switch for this conversion. Here, we attached the environment-sensitive fluorophore acrylodan (Acr) at either lobe of intact CaM or lobe-specific Ca2+-sensitive CaM mutants, and monitored the effects of Ca2+ binding via the fluorescence change of free or RyR1-bound AcrCaM. Using steady state measurements, we found that Ca2+ binding to free CaM causes a dramatic structural change in the N-lobe, but only a slight effect on the C-lobe of the Ca2+-sensitive lobe-specific mutants, in addition to the previously known higher Ca2+ affinity at the C-lobe versus the N-lobe. Using stopped-flow measurements, we found ∼30x faster Ca2+ dissociation from the N- versus C-lobe, and ∼20x slower Ca2+ association to the N-lobe versus C-lobe. These Ca2+ binding properties hold for the CaM/RyR1 complex, and Ca2+ affinity is enhanced at the CaM C-lobe but decreased at the N-lobe by RyR1 binding. We propose that fast Ca2+-binding at the C-lobe of CaM initiates its inhibition to RyR1 at high [Ca2+], while slow Ca2+ binding to the N-lobe is necessary for timely enhancement of the inhibitory effect. The dysregulation of RyR1 by M124Q-CaM may be explained by the lower Ca2+ affinity versus WT-CaM, as suggested by both steady-state and transient kinetics results.

In cardiac muscle, myosin molecules exist in multiple structural states as they transit through their ATPase cycle, including an off-cycle resting or OFF-state with their catalytic heads in a folded structure known as the interacting-heads motif (IHM). The blocked head configuration (BHC) of the IHM is unusual because its light chain binding region is held in an exaggerated prestroke angle stabilized by interactions with its own S2 tail. An additional partial OFF-state, where the second head of the IHM is not folded back onto the blocked head, has been proposed, which still has the blocked head interacting with S2. Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy are thought to destabilize (decrease the population of) the OFF-states. The effects of pathogenic mutations on the folded back structural states are often studied using indirect assays, including a single-ATP turnover assay that detects the biochemical state of myosin functionally. Here, we use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification in solution of the myosin BHC state. Using the FRET sensor, we provide evidence that the myosin tail acts as an activator of the recovery stroke transition after ATP binding to poststroke state apomyosin and that BHC formation is rapid after ATP binding and depends on formation of the prestroke state. We propose that the positively charged loop 2 of the prestroke state head interacts with the Ring 2 cluster of negatively charged residues on the S2 tail to form a preBHC state that facilitates BHC state formation.

Conformational changes triggered by kinase inhibitors are a major factor driving specificity and efficacy, but few scalable methods exist for differentiating induced conformations and binding modes. Using the receptor tyrosine kinase MET, we show that three classes of inhibitors can be distinguished by their contrasting effects on static and dynamic quenching of a fluorescent dye attached to the activation loop. Quenching is mediated by tyrosine residues on the flexible activation loop, and inhibitor binding induces order in the loop, sequestering the tyrosines and differentially suppressing static and dynamic quenching in a manner that is dependent on the induced structural state. Type I MET inhibitors have a large static and moderate dynamic component, type II inhibitors have only a static component, and active-state-selective inhibitors relieve both components to similar extents. These distinct dequenching signatures allow the straightforward detection of each binding mode by using parallel steady-state and time-resolved fluorescence measurements. We show that this technique can be applied to rapidly assess the effects of resistance mutations on inhibitor binding and can report on the chemical interactions and conformational changes that drive these effects. Conservation of the three activation loop tyrosine residues across many receptor tyrosine kinases suggests that this approach has broad utility.

In high-throughput screening (HTS) assays using fluorescence lifetime (FLT)-detected FRET, we have identified compounds that allosterically modulate the pathologically leaky ryanodine receptor (RyR) calcium release channels. These compounds may prevent or reduce the elevated Ca2+ that fuels arrhythmia, heart failure, and age-related neurodegeneration. RyRs are responsible for intracellular Ca2+ release from endoplasmic/sarcoplasmic reticulum (ER/SR). The resulting [Ca2+] pulse is a signal for many cellular processes, whereas sustained elevated [Ca2+] is pathologic. Our FRET-based HTS detects the pathology-linked RyR leaky state by monitoring binding of the accessory protein calmodulin and the DPc10 peptide (corresponding to RyR2 residues 2460-2495) known to perturb interdomain interactions within RyR2. Under conditions mimicking a pathological state, we have screened a 50,000-compound chemical library to identify small-molecule modulators of RyR2 in cardiac SR membranes. This screen yielded 603 compounds that reproducibly altered FRET. Based on FRET response profiles that align with therapeutic potential, 83 of those most promising compounds were purchased and validated by FRET dose response evaluation. Focusing on ten chemical scaffolds that desirably increase A-CaM binding, six representative compounds reduced RyR2 activity as measured by [3H]ryanodine binding. Ca2+ dynamics in HEK293 cells expressing human RyR2 or in cardiomyocytes highlighted the isoxazole group of hits as potentially therapeutic by targeting the pathological RyR2 leak state.

In cardiac muscle, many myosin molecules are in a resting or OFF state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

Using fluorescence lifetime FRET-based high-throughput screening (HTS) assays, we have identified compounds that modulate the sarcoplasmic reticulum's (SR) Ca2+ gatekeeper, the ryanodine receptor (RyR) channel. Intracellular Ca2+ regulation is critical for striated muscle function, and SR Ca2+ release via opening of RyR is essential for triggering muscle contraction. Under cellular rest, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, and neurodegenerative diseases.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.