Biology subjects

Cornea, R. L.

Publications and source records attributed to Cornea, R. L..

10 recordsLinked to original sources

bioRxiv2026

Dynamic-Structure Redesign of Calmodulin Reveals Mechanistic Constraints on Ryr2 Regulation

2.Calmodulin (CaM) is a highly conserved Ca2+ sensor that regulates hundreds of cellular targets through Ca2+ -dependent conformational dynamics. Despite its central role in Ca2+ signaling and disease, its evolutionary conservation and structural flexibility have suggested that CaM is resistant to rational redesign. Here, using the cardiac Ca2+ release channel Ryanodine receptor 2 (RyR2) as a model system, we tested whether incorporating conformational dynamics into computational protein design enables functional reengineering of CaM. We first applied a static structure-based redesign to increase CaM-RyR2 affinity. Although the resulting variant bound more tightly to both the RyR2 peptide and the intact channel in vitro, it distorted peptide geometry and worsened Ca2+ leak in cardiomyocytes ex vivo. Guided by molecular dynamics simulations, we then developed a dynamic-structure redesign strategy that preserves conformational integrity while strengthening binding. The resulting CaM variant exhibited increased RyR2 affinity and reduced pathological Ca2+ leak in a disease-relevant model. These findings show that improved binding affinity alone is insufficient to enhance physiological regulation and that successful CaM redesign requires preservation of conformational dynamics. More broadly, they demonstrate that integrating conformational dynamics into protein redesign can enable functionally predictive engineering of flexible regulatory protein-protein interactions.

biophysics↗

bioRxiv2023

Kinetics and Mapping of Ca-driven Calmodulin conformations on Skeletal and Cardiac Muscle Ryanodine Receptors

Calmodulin (CaM) transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during relaxation and contraction in cardiac and skeletal muscle. However, the structural dynamics by which CaM modulates the SR Ca2+ release channel (ryanodine receptor, RyR) at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime detection of FRET between RyR-bound FKBP and CaM, we resolved different structural states of CaM and Ca-driven shifts in the conformation of CaM bound to RyR. We found that CaM becomes more compact in contracting high-Ca2+ vs. relaxing low-Ca2+. Skeletal and cardiac RyR isoforms show different CaM-RyR conformations, and binding and structural kinetics reflect functional roles. Furthermore, our FRET methods provide insight representative of physiological CaM-RyR structure, in synergy and comparison with cryo-EM models, which result from more disrupted samples. This technology will drive future studies focusing on pathologic CaM-RyR interactions, and on RyR dynamics with other important modulators.

biophysics↗

bioRxiv2023

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

biophysics↗

bioRxiv2023

ent-Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice

Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in single RyR2 channel assays, [3H]ryanodine binding assays, and in Casq2-/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single-channels and [3H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent-B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statementThe cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

pharmacology and toxicology↗

bioRxiv2023

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer (ent) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent-verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here we used fluorescence lifetime (FLT)-measurements of Forster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multi-exponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent-verticilide, yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent-verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent-verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.

biophysics↗

bioRxiv2023

FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCAs function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca2+-ATPase and Ca2+-transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

biophysics↗

bioRxiv2021

Cardiac RyR N-terminal region biosensors for FRET-based high-throughput screening

The N-terminal region (NTR) of the ryanodine receptor (RyR) calcium channels is critical to the regulation of Ca2+ release during excitation-contraction coupling. NTR hosts numerous mutations linked to skeletal and cardiac myopathies (RyR1 and RyR2, respectively), highlighting its potential as therapeutic target. Here, we labeled the NTR of mouse RyR2 at subdomains A, B, and C with donor and acceptor pairs for fluorescence resonance energy transfer (FRET), obtaining two biosensors. Using fluorescence lifetime (FLT)-detection of intramolecular FRET, we developed high-throughput screening (HTS) assays with the biosensors to identify small-molecule modulators of RyR. We screened a 1280-compound validation library and identified several hits. Hits with saturable FRET dose-response profiles, and previously unreported effects on RyR activity, were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles, to measure their effects on full-length RyR opening in its natural membrane environment. We identified three novel inhibitors of both RyR1 and RyR2, and two RyR1-selective inhibitors at nanomolar Ca2+. These compounds may function as inhibitors of leaky RyRs in muscle. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential activators of excitation-contraction coupling. These results indicate that large-scale HTS using this platform can lead to compounds with potential for therapeutic development.

biophysics↗

bioRxiv2020

The transmembrane domain of DWORF activates SERCA directly; P15 and W22 residues are essential.

The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca2+], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

biophysics↗

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↗