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We present the NetSci program-an open-source scientific software package designed for estimating mutual information (MI) between data sets using GPU acceleration and a k-nearest-neighbor algorithm. This approach significantly enhances calculation speed, achieving improvements of several orders of magnitude over traditional CPU-based methods, with data set size limits dictated only by available hardware. To validate NetSci, we accurately compute MI for an analytically verifiable two-dimensional Gaussian distribution and replicate the generalized correlation (GC) analysis previously conducted on the B1 domain of protein G. We also apply NetSci to molecular dynamics simulations of the Sarcoendoplasmic Reticulum Calcium-ATPase (SERCA) pump, exploring the allosteric mechanisms and pathways influenced by ATP and 2'-deoxy-ATP (dATP) binding. Our analysis reveals distinct allosteric effects induced by ATP compared to dATP, with predicted information pathways from the bound nucleotide to the calcium-binding domain differing based on the nucleotide involved. NetSci proves to be a valuable tool for estimating MI and GC in various data sets and is particularly effective for analyzing intraprotein communication and information transfer.

Phospholamban (PLN) is a 52 amino acid regulin that allosterically modulates the activity of the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) in the heart muscle. In its unphosphorylated form, PLN binds SERCA within its transmembrane (TM) domains, approximately 20 Å away from the Ca 2+ binding site, reducing SERCA's apparent Ca 2+ affinity (pK Ca ) and decreasing cardiac contractility. During the enzymatic cycle, the inhibitory TM domain of PLN remains anchored to SERCA, whereas its cytoplasmic region transiently binds the ATPase's headpiece. Phosphorylation of PLN at Ser16 by protein kinase A increases the affinity of its cytoplasmic domain to SERCA, weakening the TM interactions with the ATPase, reversing its inhibitory function, and augmenting muscle contractility. How the structural changes caused by pathological mutations in the PLN cytoplasmic region are transmitted to its inhibitory TM domain is still unclear. Using solid-state NMR spectroscopy and activity assays, we analyzed the structural and functional effects of a series of mutations and their phosphorylated forms located in the PLN cytoplasmic region and linked to dilated cardiomyopathy. We found that these missense mutations affect the overall topology and dynamics of PLN and ultimately modulate its inhibitory potency. Also, the changes in the TM tilt angle and cytoplasmic dynamics of PLN caused by these mutations correlate well with the extent of SERCA inhibition. Our study unveils new molecular determinants for designing variants of PLN that outcompete endogenous PLN to regulate SERCA in a tunable manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial funding, personal interests or relationships that may appear to influence the work reported in this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The natural product curcumin and some of its analogs are known inhibitors of the transmembrane enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). Despite their widespread use, the curcuminoids' binding site in SERCA and their relevant interactions with the enzyme remain elusive. This lack of knowledge has prevented the development of curcuminoids into valuable experimental tools or into agents of therapeutic value. We used the crystal structures of SERCA in its E1 conformation in conjunction with computational tools such as docking and surface screens to determine the most likely curcumin binding site, along with key enzyme/inhibitor interactions. Additionally, we determined the inhibitory potencies and binding affinities for a small set of curcumin analogs. The predicted curcumin binding site is a narrow cleft in the transmembrane section of SERCA, close to the transmembrane/cytosol interface. In addition to pronounced complementarity in shape and hydrophobicity profiles between curcumin and the binding pocket, several hydrogen bonds were observed that were spread over the entire curcumin scaffold, involving residues on several transmembrane helices. Docking-predicted interactions were compatible with experimental observations for inhibitory potencies and binding affinities. Based on these findings, we propose an inhibition mechanism that assumes that the presence of a curcuminoid in the binding site arrests the catalytic cycle of SERCA by preventing it from converting from the E1 to the E2 conformation. This blockage of conformational change is accomplished by a combination of steric hinderance and hydrogen-bond-based cross-linking of transmembrane helices that require flexibility throughout the catalytic cycle., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stefan Paula reports financial support was provided by National Institute of General Medical Sciences. Stefan Paula reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this research, we employed the alchemical double-decoupling method alongside restraining potentials, coupled with the FEPMD method, to ascertain the standard binding free energy of a drug-like molecule termed BHQ and three analogous compounds engineered with progressive addition of bulky para-alkyl groups binding to SERCA (Ca 2+ -ATPase of skeletal muscle sarcoplasmic reticulum). Integral transmembrane proteins represent crucial drug targets in numerous therapeutic interventions, presenting computational challenges due to their considerable system sizes. Our approach integrated the generalized born potential method and the spherical solvent boundary potential method, allowing us to explicitly focus on the active binding site while treating the remainder of the system implicitly. We evaluated contributions to the standard binding free energy from distinct interaction potentials: electrostatic, repulsive, dispersive, and restraining potentials, computed separately. The resulting absolute binding free energy of BHQ (11.63 kcal/mol) closely aligns with experimental measurements (10.56 kcal/mol). Notably, an accurate estimation of the absolute binding free energy was achieved for the simplest analog, created with the addition of a single para-methyl group. However, the analog with two para-methyl groups exhibited the highest binding free energy, which disagreed with experimental results. Determining the binding free energy of the BHQ analog engineered with three para-methyl groups presented challenges in convergence and resulted in the lowest free energy among the three.

The sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) transports two Ca 2+ ions per ATP hydrolyzed from the cytoplasm to the lumen. However, how the ATP hydrolysis remotely drives the Ca 2+ transport is unclear. In the SERCA1a crystal structures, the ATP hydrolysis is accompanied by the notably increasing tilting angle of the central core (CC) and the Ca 2+ transport, and the CC tilting angle dramatically decreases in the E2 to E1 transition. We demonstrated that the significantly increasing tilting motion of the CC drove the Ca 2+ release in the molecular dynamics simulation of the R836A variant, and the dramatic spontaneous decrease in the CC tilting angle of the E2 state triggers the restart of the SERCA1a's transport cycle. The repulsion between the phosphorylated D351 and the phosphate groups in ADP triggers the release of ADP from the SERCA1a headpiece. We proposed a novel SERCA transport mechanism in which ATP hydrolysis drives a significant tilting motion of the CC, which drives Ca 2+ transport and the A domain rotational motion in the E1P-ADP-2Ca 2+ to E2P transition. The dramatic spontaneous decrease in the CC tilting angle of the E2 state drives the restart of the transport cycle., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca 2+ sensitivity of active SERCA, increasing the Ca 2+ concentration required for pump cycling. However, PLB does not decrease Ca 2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca 2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca 2+ binding, altering transport Ca 2+ dependence without actually affecting the equilibrium binding affinity of the Ca 2+ -coordinating sites. Here, we consider another hypothesis, that measurements of Ca 2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca 2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca 2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca 2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca 2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca 2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca 2+ -binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca 2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Skeletal muscles contain large numbers of high-molecular-mass protein complexes in elaborate membrane systems. Integral membrane proteins are involved in diverse cellular functions including the regulation of ion handling, membrane homeostasis, energy metabolism and force transmission., Areas Covered: The proteomic profiling of membrane proteins and large protein assemblies in skeletal muscles are outlined in this article. This includes a critical overview of the main biochemical separation techniques and the mass spectrometric approaches taken to study membrane proteins. As an illustrative example of an analytically challenging large protein complex, the proteomic detection and characterization of the Ca 2+ -ATPase of the sarcoplasmic reticulum is discussed. The biological role of this large protein complex during normal muscle functioning, in the context of fiber type diversity and in relation to mechanisms of physiological adaptations and pathophysiological abnormalities is evaluated from a proteomics perspective., Expert Opinion: Mass spectrometry-based muscle proteomics has decisively advanced the field of basic and applied myology. Although it is technically challenging to study membrane proteins, innovations in protein separation methodology in combination with sensitive mass spectrometry and improved systems bioinformatics has allowed the detailed proteomic detection and characterization of skeletal muscle membrane protein complexes, such as Ca 2+ -pump proteins of the sarcoplasmic reticulum.

Protein post-translational modifications (PTMs) are important regulators of protein functions and produce proteome complexity. SIRT1 has NAD + -dependent deacylation of acyl-lysine residues. The present study aimed to explore the correlation between lysine crotonylation (Kcr) on cardiac function and rhythm in Sirt1 cardiac-specific knockout (ScKO) mice and related mechanism. Quantitative proteomics and bioinformatics analysis of Kcr were performed in the heart tissue of ScKO mice established with a tamoxifen-inducible Cre-loxP system. The expression and enzyme activity of crotonylated protein were assessed by western blot, co-immunoprecipitation, and cell biology experiment. Echocardiography and electrophysiology were performed to investigate the influence of decrotonylation on cardiac function and rhythm in ScKO mice. The Kcr of SERCA2a was significantly increased on Lys120 (1.973 folds). The activity of SERCA2a decreased due to lower binding energy of crotonylated SERCA2a and ATP. Changes in expression of PPAR-related proteins suggest abnormal energy metabolism in the heart. ScKO mice had cardiac hypertrophy, impaired cardiac function, and abnormal ultrastructure and electrophysiological activities. We conclude that knockout of SIRT1 alters the ultrastructure of cardiac myocytes, induces cardiac hypertrophy and dysfunction, causes arrhythmia, and changes energy metabolism by regulating Kcr of SERCA2a. These findings provide new insight into the role of PTMs in heart diseases., Competing Interests: Declaration of competing interest Authors have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Marine cyanobacteria are a rich source of bioactive natural products. Here, we report the isolation and structure elucidation of the previously reported iezoside ( 1 ) and its C-31 O-demethyl analogue, iezoside B ( 2 ), from a cyanobacterial assemblage collected at Loggerhead Key in the Dry Tortugas, Florida. The two compounds have a unique skeleton comprised of a peptide, a polyketide and a modified sugar unit. The compounds were tested for cytotoxicity and effects on intracellular calcium. Both compounds exhibited cytotoxic activity with an IC 50 of 1.5 and 3.0 μΜ, respectively, against A549 lung carcinoma epithelial cells and 1.0 and 2.4 μΜ against HeLa cervical cancer cells, respectively. In the same cell lines, compounds 1 and 2 show an increase in cytosolic calcium with approximate EC 50 values of 0.3 and 0.6 μΜ in A549 cells and 0.1 and 0.5 μΜ, respectively, in HeLa cells, near the IC 50 for cell viability, suggesting that the increase in cytosolic calcium is functionally related to the cytotoxicity of the compounds and consistent with their activity as SERCA (sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase) inhibitors. The structure-activity relationship provides evidence that structural changes in the sugar unit may be tolerated, and the activity is tunable. This finding has implications for future analogue synthesis and target interaction studies.

Myoregulin (MLN) is a member of the regulin family, a group of homologous membrane proteins that bind to and regulate the activity of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA). MLN, which is expressed in skeletal muscle, contains an acidic residue in its transmembrane domain. The location of this residue, Asp35, is unusual because the relative occurrence of aspartate is very rare (<0.2%) within the transmembrane helix regions. Therefore, we used atomistic simulations and ATPase activity assays of protein co-reconstitutions to probe the functional role of MLN residue Asp35. These structural and functional studies showed Asp35 has no effects on SERCA's affinity for Ca 2+ or the structural integrity of MLN in the lipid bilayer. Instead, Asp35 controls SERCA inhibition by populating a bound-like orientation of MLN. We propose Asp35 provides a functional advantage over other members of the regulin family by populating preexisting MLN conformations required for MLN-specific regulation of SERCA. Overall, this study provides new clues about the evolution and functional divergence of the regulin family and offers novel insights into the functional role of acidic residues in transmembrane protein domains.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.

Sarco/endoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b), a member of the SERCA family, is expressed ubiquitously and transports Ca 2+ into the sarco/endoplasmic reticulum using the energy provided by ATP binding and hydrolysis. The crystal structure of SERCA2b in its Ca 2+ - and ATP-bound (E1∙2Ca 2+ -ATP) state and cryo-electron microscopy (cryo-EM) structures of the protein in its E1∙2Ca 2+ -ATP and Ca 2+ -unbound phosphorylated (E2P) states have provided essential insights into how the overall conformation and ATPase activity of SERCA2b is regulated by the transmembrane helix 11 and the subsequent luminal extension loop, both of which are specific to this isoform. More recently, our cryo-EM analysis has revealed that SERCA2b likely adopts open and closed conformations of the cytosolic domains in the Ca 2+ -bound but ATP-free (E1∙2Ca 2+ ) state, and that the closed conformation represents a state immediately prior to ATP binding. This review article summarizes the unique mechanisms underlying the conformational and functional regulation of SERCA2b., (© 2022 The Authors. BioEssays published by Wiley Periodicals LLC.)

In this issue of Structure, Reddy et al. present details about the structure, topology, and dynamics of the small membrane protein DWORF, a regulin that activates the Ca 2+ pump SERCA. State-of-the art oriented solid-state NMR spectroscopy in combination with molecular dynamics simulations reveal the structure of this cardiac muscle protein., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)