Search

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.)

Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca 2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca 2+ state, revealing a new conformation for Ca 2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca 2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca 2+ state are located at similar positions to those in the E1·2Ca 2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca 2+ . We propose a novel mechanism of ATP binding to SERCA2b., (© 2021 The Authors.)

Many bacteria export intracellular calcium using active transporters homologous to the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA). Here we present three crystal structures of Ca 2+ -ATPase 1 from Listeria monocytogenes (LMCA1). Structures with BeF 3 - mimicking a phosphoenzyme state reveal a closed state, which is intermediate between the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA. It suggests that LMCA1 in the E2P state is pre-organized for dephosphorylation upon Ca 2+ release, consistent with the rapid dephosphorylation observed in single-molecule studies. An arginine side-chain occupies the position equivalent to calcium binding site I in SERCA, leaving a single Ca 2+ binding site in LMCA1, corresponding to SERCA site II. Observing no putative transport pathways dedicated to protons, we infer a direct proton counter transport through the Ca 2+ exchange pathways. The LMCA1 structures provide insight into the evolutionary divergence and conserved features of this important class of ion transporters., 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 © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) transports Ca 2+ ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca 2+ translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa 2 has not been solved. Here, the angle of the A-domain was measured by defocused orientation imaging using isotropic total internal reflection fluorescence microscopy. A single SERCA1a molecule, labeled with fluorophore ReAsH on the A-domain in fixed orientation, was embedded in a nanodisc, and stabilized on Ni-NTA glass. Activation with ATP and Ca 2+ caused angle changes of the fluorophore and therefore the A-domain, motions lost by inhibitor, thapsigargin. Our high-speed set-up captured the motion during EP isomerization, and suggests that the A-domain rapidly rotates back and forth from an E1PCa 2 position to a position close to the E2P state. This is the first report of the detection in the movement of the A-domain as an angle change. Our method provides a powerful tool to investigate the conformational change of a membrane protein in real-time.

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium., Competing Interests: MF, EB, RA, EC, MP, MD, NR, LE, SR, AZ, HY No competing interests declared, ML Reviewing editor, eLife, (© 2021, Fisher et al.)

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca 2+ -transport response to β-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 Ca 2+ -ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca 2+ 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 PLN's 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 enzyme's active site, augmenting Ca 2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins., Competing Interests: DW, UR, SW, EL, TG, MG, RC, DT, AD, GV No competing interests declared, (© 2021, Weber et al.)

The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with calcium ATPase from sarcoplasmic reticulum (SR) was studied by means of different approaches, such as enzyme activity, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and molecular docking simulations. The ATP hydrolysis activity was fully inhibited by incubation with dirhamnolipid (diRL) up to 0.1 mM concentration, corresponding to a surfactant concentration below membrane solubilization threshold. Surfactant-protein interaction induced conformational changes in the protein observed by an increase in the accessibility of tryptophan residues to the aqueous phase and by changes in the secondary structure of the protein as seen by fluorescence and FTIR spectroscopy. As a consequence, the protein become more unstable and denatured at lower temperatures, as seen by enzyme activity and DSC studies. Finally, these results were explained at molecular level throughout molecular docking simulations. It is concluded that there is a specific dirhamnolipid-protein interaction not related to the surface activity of the surfactant but to the particular physicochemical properties of the biosurfactant molecule., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca 2+ signal is well-known to trigger a cascade of events leading to altered response to variety of stimuli and persistent accumulation of pathological changes at the molecular level. To counterbalance potentially detrimental consequences of Ca 2+ , neurons are equipped with sophisticated mechanisms that function to keep its concentration in a tightly regulated range. Calcium pumps belonging to the P-type family of ATPases: plasma membrane Ca 2+ -ATPase (PMCA), sarco/endoplasmic Ca 2+ -ATPase (SERCA) and secretory pathway Ca 2+ -ATPase (SPCA) are considered efficient line of defense against abnormal Ca 2+ rises. However, their role is not limited only to Ca 2+ transport, as they present tissue-specific functionality and unique sensitive to the regulation by the main calcium signal decoding protein-calmodulin (CaM). Based on the available literature, in this review we analyze the contribution of these three types of Ca 2+ -ATPases to neuropathology, with a special emphasis on mental diseases.

Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA1a). Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca 2 .E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.

The endoplasmic reticulum (ER) contains various enzymes that metabolize fatty acids (FAs). Given that FAs are the components of membranes, FA metabolic enzymes might be associated with regulation of ER membrane functions. However, it remains unclear whether there is the interplay between FA metabolic enzymes and ER membrane proteins. Trans-2-enoyl-CoA reductase (TER) is an FA reductase present in the ER membrane and catalyzes the last step in the FA elongation cycle and sphingosine degradation pathway. Here we identify sarco(endo)plasmic reticulum Ca 2+ -ATPase 2b (SERCA2b), an ER Ca 2+ pump responsible for Ca 2+ accumulation in the ER, as a TER-binding protein by affinity purification from HEK293 cell lysates. We show that TER directly binds to SERCA2b by in vitro assays using recombinant proteins. Thapsigargin, a specific SERCA inhibitor, inhibits this binding. TER binds to SERCA2b through its conserved C-terminal region. TER overexpression suppresses SERCA2b ATPase activity in microsomal membranes of HEK293 cells. Depletion of TER increases Ca 2+ storage in the ER and accelerates SERCA2b-dependent Ca 2+ uptake to the ER after ligand-induced Ca 2+ release. Moreover, depletion of TER reduces the Ca 2+ -dependent nuclear translocation of nuclear factor of activated T cells 4. These results demonstrate that TER is a negative regulator of SERCA2b, implying the direct linkage of FA metabolism and Ca 2+ accumulation in the ER., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regards to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Helical assemblies of proteins, which consist of a two-dimensional lattice of identical subunits arranged with helical symmetry, are a common structural motif in nature. For membrane proteins, crystallization protocols can induce helical arrangements and take advantage of the symmetry found in these assemblies for the structural determination of target proteins. Modern advances in the field of electron cryo-microscopy (cryo-EM), in particular the advent of direct electron detectors, have opened the potential for structure determination of membrane proteins in such assemblies at high resolution. The nature of the symmetry in helical crystals of membrane proteins means that a single image potentially contains enough information for three-dimensional structural determination. With the current direct electron detectors, we have never been closer to making this a reality. Here, we present a protocol detailing the preparation of helical crystals, with an emphasis on further cryo-EM analysis and structural determination of the sarco(endo)plasmic reticulum Ca 2+ -ATPase in the presence of regulatory subunits such as phospholamban.

The sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) is a P-type ATPase that transports Ca 2+ from the cytosol into the sarco(endo)plasmic reticulum (SR/ER) lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca 2+ -binding sites and the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of 5 of SERCA's 10 transmembrane helices. The mutant displays a marked slowing of the Ca 2+ -binding kinetics, and its crystal structure in the presence of Ca 2+ and ATP analog reveals a rotated headpiece, altered connectivity between the cytosolic domains, and an altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca 2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in interdomain communication between the headpiece and the Ca 2+ -binding transmembrane region., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) and phospholamban (PLB) are essential components of the cardiac Ca 2+ transport machinery. PLB phosphorylation at residue Ser16 (pSer16) enhances SERCA activity in the heart via an unknown structural mechanism. Here, we report a fully atomistic model of SERCA bound to phosphorylated PLB and study its structural dynamics on the microsecond time scale using all-atom molecular dynamics simulations in an explicit lipid bilayer and water environment. The unstructured N-terminal phosphorylation domain of PLB samples different orientations and covers a broad area of the cytosolic domain of SERCA but forms a stable complex mediated by pSer16 interactions with a binding site formed by SERCA residues Arg324/Lys328. PLB phosphorylation does not affect the interaction between the transmembrane regions of the two proteins; however, pSer16 stabilizes a disordered structure of the N-terminal phosphorylation domain that releases key inhibitory contacts between SERCA and PLB. We found that PLB phosphorylation is sufficient to guide the structural transitions of the cytosolic headpiece that are required to produce a competent structure of SERCA. We conclude that PLB phosphorylation serves as an allosteric molecular switch that releases inhibitory contacts and strings together the catalytic elements required for SERCA activation. This atomistic model represents a vivid atomic-resolution visualization of SERCA bound to phosphorylated PLB and provides previously inaccessible insights into the structural mechanism by which PLB phosphorylation releases SERCA inhibition in the heart.

Under physiological conditions, most Ca 2+ -ATPase (SERCA) molecules bind ATP before binding the Ca 2+ transported. SERCA has a high affinity for ATP even in the absence of Ca 2+ , and ATP accelerates Ca 2+ binding at pH values lower than 7, where SERCA is in the E2 state with low-affinity Ca 2+ -binding sites. Here we describe the crystal structure of SERCA2a, the isoform predominant in cardiac muscle, in the E2·ATP state at 3.0-Å resolution. In the crystal structure, the arrangement of the cytoplasmic domains is distinctly different from that in canonical E2. The A-domain now takes an E1 position, and the N-domain occupies exactly the same position as that in the E1·ATP·2Ca 2+ state relative to the P-domain. As a result, ATP is properly delivered to the phosphorylation site. Yet phosphoryl transfer never takes place without the filling of the two transmembrane Ca 2+ -binding sites. The present crystal structure explains what ATP binding itself does to SERCA and how nonproductive phosphorylation is prevented in E2., Competing Interests: The authors declare no competing interest.

P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H + , Na + , K + , Ca 2+ , and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca 2+ -ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.

Sarcoendoplasmic reticulum calcium ATPase (SERCA), a member of the P-type ATPase family of ion and lipid pumps, is responsible for the active transport of Ca 2+ from the cytoplasm into the sarcoplasmic reticulum lumen of muscle cells, into the endoplasmic reticulum (ER) of non-muscle cells. X-ray crystallography has proven to be an invaluable tool in understanding the structural changes of SERCA, and more than 70 SERCA crystal structures representing major biochemical states (defined by bound ligand) have been deposited in the Protein Data Bank. Consequently, SERCA is one of the best characterized components of the calcium transport machinery in the cell. Emerging approaches in the field, including spectroscopy and molecular simulation, now help integrate and interpret this rich structural information to understand the conformational transitions of SERCA that occur during activation, inhibition, and regulation. In this review, we provide an overview of the crystal structures of SERCA, focusing on identifying metrics that facilitate structure-based categorization of major steps along the catalytic cycle. We examine the integration of crystallographic data with different biophysical approaches and computational methods to link biochemical and structural states of SERCA that are populated in the cell. Finally, we discuss the challenges and new opportunities in the field, including structural elucidation of functionally important and novel regulatory complexes of SERCA, understanding the structural basis of functional divergence among homologous SERCA regulators, and bridging the gap between basic and translational research directed toward therapeutic modulation of SERCA.

Sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca 2 ]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca 2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The sarco/endoplasmic reticulum calcium ATPase (SERCA), which plays a key role in the maintenance of Ca 2+ ion homeostasis, is an extensively studied enzyme, the inhibition of which has a considerable impact on cell life and death decision. To date, several SERCA inhibitors have been thoroughly studied and the most notable one, a derivative of the sesquiterpene lactone thapsigargin, is gradually approaching a clinical application. Meanwhile, new compounds with SERCA-inhibiting properties of natural, synthetic, or semisynthetic origin are being discovered and/or developed; some of these might also be suitable for the development of new drugs with improved performance. This review brings an up-to-date comprehensive overview of recently discovered compounds with the potential of SERCA inhibition, discusses their mechanism of action, and highlights their potential clinical applications, such as cancer treatment.

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na + ,K + -, H + ,K + - and sarcoplasmic reticulum Ca 2+ -ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na + ,K + - and H + ,K + -ATPases compared to the Ca 2+ -ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na + ,K + -ATPase and the Ca 2+ -ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na + ,K + -ATPase than the E2 conformation, but in the case of the Ca 2+ -ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na + ,K + - and H + ,K + -ATPases, the E2-E1 transition of the Ca 2+ -ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na + ,K + -ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na + , K + - and the H + ,K + -ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) maintains the level of calcium concentration in cells by pumping calcium ions from the cytoplasm to the lumen while undergoing substantial conformational changes, which can be stabilized or prevented by various compounds. Here we attempted to clarify the molecular mechanism of action of new inhibitor rutin arachidonate, one of the series of the acylated rutin derivatives. We performed molecular dynamics simulations of SERCA1a protein bound to rutin arachidonate positioned in a pure dipalmitoylphosphatidylcholine bilayer membrane. Our study predicted the molecular basis for the binding of rutin arachidonate towards SERCA1a in the vicinity of the binding site of calcium ions and near the location of the well-known inhibitor thapsigargin. The stable hydrogen bond between Glu771 and rutin arachidonate plays a key role in the binding. SERCA1a is kept in the E2 conformation preventing the formation of important salt bridges between the side chains of several residues, primarily Glu90 and Lys297. All in all, the structural changes induced by the binding of rutin arachidonate to SERCA1a may shift proton balance near the titrable residues Glu771 and Glu309 into neutral species, hence preventing the binding of calcium ions to the transmembrane binding sites and thus affecting calcium homeostasis. Our results could lead towards the design of new types of inhibitors, potential drug candidates for cancer treatment, which could be anchored to the transmembrane region of SERCA1a by a lipophilic fatty acid group., Competing Interests: The authors declare no competing conflict of interest.

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn 4 -Ala and Thr 5 -Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Membrane transporters contribute to the regulation of the internal environment of cells by translocating substrates across cell membranes. Like all physical systems, the behaviour of membrane transporters is constrained by the laws of thermodynamics. However, many mathematical models of transporters, especially those incorporated into whole-cell models, are not thermodynamically consistent, leading to unrealistic behaviour. In this paper we use a physics-based modelling framework, in which the transfer of energy is explicitly accounted for, to develop thermodynamically consistent models of transporters. We then apply this methodology to model two specific transporters: the cardiac sarcoplasmic/endoplasmic Ca 2+ ATPase (SERCA) and the cardiac Na + /K + ATPase., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA. In view of these two alternative binding interactions, we evaluated the possibility that oligomerization occurs at the expense of SERCA-binding. However, even the most avidly oligomeric micropeptide species still showed robust FRET with SERCA, and there was a surprising positive correlation between oligomerization affinity and SERCA-binding. This comparison of micropeptide family members suggests that the same structural determinants that support oligomerization are also important for binding to SERCA. Moreover, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory function of the other SERCA-binding micropeptides., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The capability to handle highly heterogeneous molecular assemblies in a consistent manner is among the greatest challenges faced when deriving simulation parameters. This is particularly the case for coarse-grained (CG) simulations in which chemical functional groups are lumped into effective interaction centers for which transferability between different chemical environments is not guaranteed. Here, we introduce the parametrization of a set of CG phospholipids compatible with the latest version of the SIRAH force field for proteins. The newly introduced lipid species include different acylic chain lengths and partial unsaturation, as well as polar and acidic head groups that show a very good reproduction of structural membrane determinants, such as areas per lipid, thickness, order parameter, etc., and their dependence with temperature. Simulation of membrane proteins showed unprecedented accuracy in the unbiased description of the thickness-dependent membrane-protein orientation in systems where this information is experimentally available (namely, the SarcoEndoplasmic Reticulum Calcium-SERCA-pump and its regulator Phospholamban). The interactions that lead to this faithful reproduction can be traced down to the single amino acid-lipid interaction level and show full agreement with biochemical data present in the literature. Finally, the present parametrization is implemented in the GROMACS and AMBER simulation packages facilitating its use by a wide portion of the biocomputing community.

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

The knowledge about the role of calcium-regulated pathways in cancer cell growth and differentiation could be useful for the development of new therapeutic approaches to diminish its mortality. The ATP2A genes encode for SERCA pumps, which modulate cytosolic Ca 2+ concentration, regulating various cellular processes including cell growth. ATP2A3 gene transcriptional down-regulation has been reported in gastric and colon cancer, but there is still a lack of understanding about the epigenetic processes regulating its transcription. In this work, we report that butyrate, trichostatin A, and 5-azacytidine treatments increase SERCA3 expression, increased apoptosis, and decreased cell viability of the KATO-III gastric carcinoma cell line. We analyzed the methylation profile of the ATP2A3 gene promoter CpG island, finding clones with methylated status through -280 to -135 promoter region, harboring Sp1 and AP-2 binding sites, which could have a role in transcriptional repression. Post-translational modifications of histones show a major role in the ATP2A3 transcriptional regulation, and our results show histones marks linked to transcriptional repression associated with the -262 to -135 region, this repressive context changed to transcriptional permissive through SERCA3 re-expressing conditions. These results suggest that the nucleotide sequence from -280 to -135 position is an ATP2A3 epigenetic regulatory CpG region in KATO-III cells. Analyses of online-databases show a decreased SERCA3 expression in gastric and colon tumors, as well as overall survival results, showed that high SERCA3 expression could serve as a favorable prognostic marker for colon and gastric cancer patients., (© 2019 Wiley Periodicals, Inc.)

Carazolol (CZL) is a known agonist of β3 and antagonist of β1 and β2 adrenoceptors (AR), used in the animal production industry to improve meat quality by reducing animal stress and skeletal muscle (SM) proteolysis. Here we sought to better understand the direct effect CZL has on SM. We study CZL effect on calcium (Ca 2+ ) regulation by enzymatic activity kinetics of the Ca 2+ -ATPase (SERCA), in isolated sarcoplasmic reticulum (SR) from SM and on the mechanical properties of isolated muscle. In isolated SR from SM previously incubated with 0.03 mM CZL, but absent during SR isolation and during SERCA activity determination, the activity was reduced by 45%. Thermal analysis of SERCA activity with CZL shifted the transition temperature of inactivation (T i ) from T i  = 47 to 44 °C. When isolated SR from fast and slow SM was exposed to CZL, inhibition of SERCA occurred in a dose dependent manner. Slow and fast SM T i of SERCA shifted to a lower temperature in the presence of CZL and a second transition appears at temperatures <40 °C. In isolated extensor digitorum longus (EDL) and soleus muscles, CZL reduces the contraction force and increases susceptibility to fatigue. However, recovery force after fatigue in either muscle was higher. Our results suggest that Carazolol penetrates the plasma membrane and interacts with SERCA, thus having an important effect on skeletal muscle function. The inhibition of SERCA may lead to a decrement in SR Ca 2+ -release promoting further failure in muscle contraction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca 2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11 th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca 2+ -adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and the N-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca 2+ ) binds SERCA with micromolar affinity, magnesium (Mg 2+ ) and potassium (K + ) also compete with Ca 2+ binding. However, the molecular bases for these competing ions' influence on the SERCA function and the selectivity of the pump for Ca 2+ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca 2+ binding sites via (1) triplicate molecular dynamics (MD) simulations of Mg 2+ , Ca 2+ , and K + -bound SERCA, (2) mean spherical approximation (MSA) theory to score 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 is optimized for Ca 2+ and to a lesser extent for Mg 2+ and K + , as determined by MD-derived cation-amino acid oxygen and bound water configurations, (b) the impaired coordination and high desolvation cost for Mg 2+ precludes favorable Mg 2+ binding relative to Ca 2+ , while K + has limited capacity to bind site I, and (c) Mg 2+ most likely acts as inhibitor and K + as intermediate in SERCA's 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 rationalizes enzyme activity across broad ranges of K + , Ca 2+ , and Mg 2+ concentrations.

The calcium pump SERCA is a transmembrane protein that is critical for calcium transport in cells. SERCA resides in an environment made up largely by the lipid bilayer, so lipids play a central role on its stability and function. Studies have provided insights into the effects of annular and bulk lipids on SERCA activation, but the role of a nonannular lipid site in the E2 intermediate state remains elusive. Here, we have performed microsecond molecular dynamics simulations to probe the effects of nonannular lipid binding on the stability and structural dynamics of the E2 state of SERCA. We found that the structural integrity and stability of the E2 state is independent of nonannular lipid binding, and that occupancy of a lipid molecule at this site does not modulate destabilization of the E2 state, a step required to initiate the transition toward the competent E1 state. We also found that binding of the nonannular lipid does not induce direct allosteric control of the intrinsic functional dynamics the E2 state. We conclude that nonannular lipid binding is not necessary for the stability of the E2 state, but we speculate that it becomes functionally significant during the E2-to-E1 transition of the pump.

The sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca 2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3 Å) and the Ca 2+ -occluded [Ca 2 ]E1-AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a., (© 2019 The Authors.)

The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Solid-state NMR (ssNMR) is an ideal tool to study structure and dynamics of membrane proteins in their native lipid environment. In principle, ssNMR has no size limitations. However, this feature is rarely exploited as large membrane proteins display severe resonance overlap. In addition, dismal yields from recombinant bacterial expression systems limit severely spectroscopic characterization of membrane proteins. For very large mammalian membrane proteins, extraction from the original organism remains the most viable approach. In this case, NMR-observable nuclei must be introduced post-translationally, but the approaches developed so far are rather scarce. Here, we detail the synthesis and engineering of a reactive 13 C-ethylmethanethiosulfonate ( 13 C-EMTS) reagent for the post-translational alkylation of cysteine sidechains of a 110kDa sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) extracted from rabbit skeletal muscle tissue. When reconstituted into liposomes, it is possible to resolve the resonances of the engineered ethyl groups by magic-angle spinning (MAS) 2D [ 13 C, 13 C]-DARR experiments. Notably, the ethyl-group modification does not perturb the function of SERCA, yielding well-resolved 13 C- 13 C fingerprints that are used to image its structural states in the catalytic cycle and filtering out overwhelming naturally-abundant 13 C nuclei signals arising from the enzyme and lipids. We anticipate that this approach will be used together with 19 F NMR to monitor conformational transitions of enzymes and proteins that are difficult to express recombinantly., (© 2019 Elsevier Inc. All rights reserved.)

Ca 2+ -ATPase of sarcoplasmic reticulum (SERCA1a) pumps two Ca 2+ per ATP hydrolyzed from the cytoplasm and two or three protons in the opposite direction. In the E2 state, after transferring Ca 2+ into the lumen of sarcoplasmic reticulum, all of the acidic residues that coordinate Ca 2+ are thought to be protonated, including the gating residue Glu309. Therefore a Glu309Gln substitution is not expected to significantly perturb the structure. Here we report crystal structures of the Glu309Gln and Glu309Ala mutants of SERCA1a under E2 conditions. The Glu309Gln mutant exhibits, unexpectedly, large structural rearrangements in both the cytoplasmic and transmembrane domains, apparently uncoupling them. However, the structure definitely represents E2 and, together with the help of quantum chemical calculations, allows us to postulate a mechanism for the E2 → E1 transition triggered by deprotonation of Glu309., Competing Interests: The authors declare no conflict of interest.

The calcium pump of the sarcoplasmic reticulum (SERCA) is an ATP-driven active transporter of Ca 2+ ions that functions via an "alternating-access" cycle mechanism. In each cycle, SERCA transports two Ca 2+ ions toward the lumen of the sarcoplasmic reticulum and two to three protons to the cytoplasm. How the latter conformational transition is coupled to cytoplasmic release of protons remains poorly understood. The present computational study shows how the mechanism of proton countertransport is coupled to the alternating access gating process in SERCA. Molecular dynamics simulation trajectories are generated starting from a series of configurations taken along the E2 to E1 transition pathway determined by the string method with swarms-of-trajectories. Simulations of different protonation configurations at the binding sites reveal how deprotonation events affect the opening of the cytoplasmic gate. The results show that there is a strong coupling between the chronological order of deprotonation, the entry of water molecules into the TM region, and the opening of the cytoplasmic gate. Deprotonation of E309 and E771 is sequential with E309 being the first to lose the proton. The deprotonation promotes the opening of the cytoplasmic gate but leads to a productive gating transition only if it occurs after the transmembrane domain has reached an intermediate conformation. Deprotonation of E309 and E771 is unproductive when it occurs too early because it causes the re-opening of the luminal gate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)