Biology subjects

Kekenes-Huskey, P. M.

Publications and source records attributed to Kekenes-Huskey, P. M..

11 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↗

bioRxiv2025

Prediction of biomolecule kinetics using physics-based Brownian dynamics to data-driven machine learning methods

1We review the Brownian dynamics (BD) modeling of biomolecular binding events, with an emphasis on enzyme-substrate interactions in cellular environments. We begin with theoretical foundations of BD and its applications to association and dissociation binding processes in both homogeneous and heterogeneous media. Next, we discuss BD in the context of continuum methods and emerging machine learning (ML) approaches toward predicting binding kinetics in vivo, with an eye toward multiscale modeling perspectives. Finally, we position BD simulations as a bridge to couple atomistic-scale models with dynamic, systems biology-based descriptions of cellular processes as a final frontier in modeling target/enzyme binding processes.

biophysics↗

bioRxiv2023

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

physiology↗

bioRxiv2021

STOICHIOMETRY OF THE SODIUM PUMP-PHOSPHOLEMMAN REGULATORY COMPLEX

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.

biophysics↗

bioRxiv2021

Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia

1Adenosine triphosphate (ATP) drives microglia motility and cytokine production by activating P2X- and P2Y- class purinergic receptors with extracellular ATP and its metabolites. Purinergic receptor activation gives rise to diverse intracellular Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these diverse waveforms influence microglia function is not well established. We developed a computational model trained with published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration and how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced calcium waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. We, therefore, next evaluated how purinoreceptors and ectonucleotidase work in tandem. Our modeling results indicate that small transients are sufficient to promote motility, while large and sustained transients are needed for cytokine responses. Lastly, we predict how these phenotypical responses vary in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model can be extrapolated to diverse microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence the microglias responses to ATP.

physiology↗

bioRxiv2021

The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin.

Calmodulin (CaM) is an important Ca2+ signaling hub that regulates many protein signaling pathways. In recent years, several CaM homologs expressed in plants have been shown to regulate mammalian targets and they are attractive for gene therapy. However, the molecular basis of how the CaM homologs mutations impact target activation is unclear, which limits efforts to engineer their functional properties. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isofroms, sCaM-1 and sCaM-4 share >90% and ~ 78% identity with the mammalian CaM (mCaM), respectively, activate CaN with comparable or reduced activity relative to mCaM. We used molecular simulations and experimental assays to probe whether calcium and protein-protein binding interactions are altered in plant CaMs relative to mCaM as a basis for differential CaN regulations. We found that the two sCaMs Ca2+-binding properties such as coordination and affinity are comparable to mCaM. Further, the binding of CaM to the CaM binding region (CaMBR) in CaN is also comparable among the three CaMs, as evidenced by calculated binding free energies and experimental measured EC50 [CaM]. However, mCaM and sCaM-1 exhibited stronger binding with a secondary region of CaNs regulatory domain that is weakened for sCaM-4. This secondary interaction is likely to affect the turnover rate (kcat) of CaN based on our modeling of enzyme activity and is consistent with our experimental data. Together, our data show how plant-derived CaM variants can alter target activation through interactions beyond Ca2+-binding and canonical CaMBR binding, which may extend beyond the mammalian CaN target.

biochemistry↗

bioRxiv2021

Assessing the Role of CaM's Linker Flexibility in Target Binding

1Calmodulin (CaM) is a universal Ca2+ binding protein known to bind at least 300 targets. The selectivity and specificity towards these targets are partially attributed to the proteins flexible alpha-helical linker that connects its N- and C-domains. How this flexible linker mediates the driving forces guiding CaMs binding to regulatory targets is not well-established. Therefore, we utilized the Martini coarse-grained (CG) molecular dynamics simulations to probe interrelationships between CaM/target assembly and the role of its linker region. As a model system, we simulated the binding of CaM to the CaM binding region (CaMBR) of calcineurin (CaN). The simulations were conducted assuming a wild-type calmodulin with normal flexibility of its linker, as well as a labile, highly flexible linker variant. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 {micro}s of simulation time; of these, 1.7% sampled the fully-bound state observed in experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be ka = 5.9 x 108 M-1 s-1, which is similar to the experimentally-determined rate of 2.2 x 108 M-1 s-1 [1]. Further, our simulations recapitulated the inverse relationship between the association rate and solution ionic strength reported in the literature. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states controlled by the linker properties. Specifically, the labile linker variant samples fewer extended states compatible with target peptide binding. Therefore, our simulations suggest that variations in the CaM linkers propensity for alpha-helical secondary structure can modulate the kinetics of target binding. This finding is important, as the linker region houses several CaM variants sites for post-translational modifications, that may alter the proteins normal regulatory functions.

biophysics↗

bioRxiv2019

Co-localization and confinement of ecto-nucleotidases modulate extracellular adenosine nucleotide pools

1Nucleotides comprise small molecules that perform critical signaling and energetic roles in biological systems. Of these, the concentrations of adenosine and its derivatives, including adenosine tri-, di-, and mono-phosphate are dynamically controlled in the extracellular-space by diphosphohydrolases and ecto-nucleotidases that rapidly degrade such nucleotides. In many instances, the close coupling between cells such as those in synaptic junctions yields tiny extracellular nanodomains, within which the charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of sequentially-coupled diphosphohydrolase/nucleotidase-catalyzed reactions. To rank the relative importance of these factors, we utilize reaction-diffusion numerical simulations to systematically probe coupled enzyme activity in narrow junctions. We perform these simulations in nanoscale geometries representative of narrow extracellular compartments, within which we localize sequentially- and spatially-coupled enzymes. These enzymes catalyze the conversion of a representative charged substrate such as adenosine triphosphate (ATP) into substrates with different net charges, such as adenosine monophosphate (AMP) and adenosine (Ado). Our modeling approach considers electrostatic interactions of diffusing, charged substrates with extracellular membranes, and coupled enzymes. With this model, we find that 1) Reaction rates exhibited confinement effects, namely reduced reaction rates relative to bulk, that were most pronounced when the enzyme was close to the pore size and 2) The presence of charge on the pore boundary further tunes reaction rates by controlling the pooling of substrate near the reactive protein akin to ions near trans-membrane proteins. These findings suggest how remarkable reaction efficiencies of coupled enzymatic processes can be supported in charged and spatially-confined volumes of extracellular spaces.

biophysics↗

bioRxiv2018

Cation binding to SERCA

Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca2+) binds SERCA with micro-molar affinity, magnesium (Mg2+) and potassium (K+) also compete with calcium (Ca2+) binding. However, the molecular bases for these competing ions influence on SERCA function and the selectivity of the pump for Ca2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca2+ binding sites: 1) triplicate molecular dynamics (MD) simulations of Mg2+, Ca2+and K+-bound SERCA. 2) mean spherical approximation (MSA) theory to determine the affinity and selectivity of cation binding to the MD-resolved structures and 3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that a) coordination at sites I and II are optimized for Ca2+and to a lesser extent for Mg2+ and K+, as determined by MD-derived cation-amino acid oxygen and bound water configurations, b) the impaired coordination and high desolvation cost for Mg2+ precludes favorable Mg2+ binding relative to Ca2+, while K+ has limited capacity to bind site I, c) Mg2+ most likely acts as inhibitor and K+ as intermediate in SERCAs reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalize enzyme activity across broad ranges of K+, Ca2+ and Mg2+ concentrations.

biochemistry↗

bioRxiv2018

Matched-filter based algorithm for sub- to multi-cellular classification of myocyte tubule-system

In mammalian ventricular cardiomyocytes, invaginations of the surface membrane form the transverse tubular system (T-system) which consists of transverse tubules (TTs) that align with sarcomeres and Z-lines as well as longitudinal tubules (LTs) that are present between Z-lines in some species. In many cardiac disease etiologies the T-system is perturbed, which is believed to promote spatially heterogeneous, dyssynchronous Ca2+ release and inefficient contraction. In general, T-system characterization approaches have been directed primarily at isolated cells and do not detect subcellular T-system heterogeneity. Here we present MatchedMyo, a matched-filter based algorithm for subcellular T-system characterization in isolated cardiomyocytes and millimeter-scale myocardial sections. The algorithm utilizes "filters" representative of TTs, LTs, and T-system absence. Application of the algorithm to cardiomyocytes isolated from rat disease models of myocardial infarction (MI), dilated cardiomyopathy induced via aortic banding (AB), and sham surgery confirmed and quantified heterogeneous T-system structure and remodeling. Cardiomyocytes from post-MI hearts exhibited increasing T-system disarray as proximity to the infarct increased. We found significant (p<0.05, Welchs t-test) increases in LT density within cardiomyocytes proximal to the infarct (12{+/-}3%, data reported as mean {+/-} SD, n=3) vs. sham (4{+/-}2%, n=5), but not distal to the infarct (7{+/-}1%, n=3). The algorithm also detected decreases in TTs within 5{degrees} of the myocyte minor axis for isolated AB (36{+/-}9%, n=3) and MI cardiomyocytes located intermediate (37{+/-}4%, n=3) and proximal (34{+/-}4%, n=3) to the infarct vs. sham (57{+/-}12%, n=5). Application of bootstrapping to rabbit MI tissue revealed distal sections comprised 18.9{+/-}1.0% TTs while proximal sections comprised 10.1{+/-}0.8% TTs (p<0.05), a 46.6% decrease. The matched filter approach therefore provides a robust and scalable technique for T-system characterization from isolated cells through millimeter-scale myocardial sections.

biophysics↗

bioRxiv2018

Dual roles of electrostatic-steering and conformational dynamics in the binding of calcineurin’s intrinsically-disordered recognition domain to calmodulin

calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The CaN regulatory domain (RD) is responsible for regulating the enzymes phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorderto-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating CaM binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder cofacilitate or hinder CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.

biophysics↗