Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

Satellite double-stranded RNA induces mesenchymal transition in pancreatic cancer by regulating alternative splicing.

Human satellite II (HSATII), composed of tandem repeats in pericentromeric regions, is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of repetitive elements in cancer tissues can facilitate incidental dsRNA formation; however, it remains controversial whether dsRNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the double-stranded formation of HSATII RNA and explored its molecular function. The overexpression of double-stranded HSATII (dsHSATII) RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, spermatid perinuclear RNA-binding protein (STRBP), which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. The mesenchymal transition of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with epithelial-mesenchymal transition (EMT). We also confirmed that isoform switching of CLSTN1, driven by dsHSATII overexpression or STRBP depletion, induced EMT-like morphological changes. These findings reveal a novel tumor-promoting function of dsHSATII RNA, inducing EMT-like changes and cell invasiveness, thus enhancing our understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors.

Uncovering allostery and regulation in SORCIN through molecular dynamics simulations.

Soluble resistance-related calcium-binding protein or Sorcin is an allosteric, calcium-binding Penta-EF hand (PEF) family protein implicated in multi-drug resistant cancers. Sorcin is known to bind chemotherapeutic molecules such as Doxorubicin. This study uses in-silico molecular dynamics simulations to explore the dynamics and allosteric behavior of Sorcin in the context of Ca2+ uptake and Doxorubicin binding. The results show that Ca2+ binding induces large, but reversible conformational changes in the Sorcin structure which manifest as rigid body reorientations that preserve the local secondary structure. A reciprocal allosteric handshake centered around the EF5 hand is found to be key in Sorcin dimer formation and stabilization. Binding of Doxorubicin results in rearrangement of allosteric communities which disrupts long-range allosteric information transfer from the N-terminal domain to the middle lobe. However, this binding does not result in secondary structure destabilization. Sorcin does not appear to have a distinct Ca2+ activated mode of Doxorubicin binding.Communicated by Ramaswamy H. Sarma.

Development of a fluorescent multiplexed lateral flow immunoassay for the simultaneous detection of crustacean allergen tropomyosin, sarcoplasmic calcium binding protein and egg allergen ovalbumin in different matrices and commercial foods.

A quantum dot (QD) based multiplexed lateral flow immunoassay (xLFIA) for the simultaneous detection of egg allergen ovalbumin, crustacean allergen tropomyosin (TM) and sarcoplasmic calcium binding protein (SCP) was developed in this study. QD-labeled rabbit anti-ovalbumin, SCP and TM antibodies were applied as fluorescent detection probes. The chromatography system was optimized to reduce the mutual interference of different test lines. Visual and instrumental detection limits of the xLFIA were 0.1 and 0.05 µg/mL for SCP, both 0.05 µg/mL for ovalbumin and both 0.5 µg/mL for TM. As low as 0.10 % crab powder, 0.01 % egg white powder and 0.05 % shrimp powder could be detected in all three model foods using xLFIA. Besides, the xLFIA detection results of 23 of 28 commercial foods were consistent with ingredient labels. These findings indicate that the developed xLFIA is a practical tool for point-of-care detection of egg and crustacean allergens in processed and commercial foods.

Purification, Expression, and Characterization of Sarcoplasmic Calcium Binding Protein: A Novel Allergen of Portunus trituberculatus.

Swimming crab (Portunus trituberculatus), a crucial valuable crustacean, is a common factor causing food allergy. However, studies on allergens of P. trituberculatus are scarce. In this study, the sarcoplasmic calcium binding protein (SCP) of P. trituberculatus was expressed in Escherichia coli, purified with affinity chromatography, and the IgE-binding activity was evaluated through serological analyses. Further, the structure, physicochemical properties, and cross-reactivity were assessed via bioinformatics, immunologic, and spectroscopy techniques. The results indicated that P. trituberculatus SCP was an allergen displaying strong IgE-binding capacity, composed of 60% α-helix. It presented good immunologic and structural stability at 4-70 °C and pH 3-10, and only exhibited high IgG cross-reactivity among crustaceans, without cross-reactivity with other species tested. These results establish the foundations for further studies on SCP and are promising to promote the development of specific crustacean allergen detection and precise allergy diagnosis.

The Dysferlin C2A Domain Binds PI(4,5)P2 and Penetrates Membranes.

Dysferlin is a large membrane protein found most prominently in striated muscle. Loss of dysferlin activity is associated with reduced exocytosis, abnormal intracellular Ca2+ and the muscle diseases limb-girdle muscular dystrophy and Miyoshi myopathy. The cytosolic region of dysferlin consists of seven C2 domains with mutations in the C2A domain at the N-terminus resulting in pathology. Despite the importance of Ca2+ and membrane binding activities of the C2A domain for dysferlin function, the mechanism of the domain remains poorly characterized. In this study we find that the C2A domain preferentially binds membranes containing PI(4,5)P2 through an interaction mediated by residues Y23, K32, K33, and R77 on the concave face of the domain. We also found that subsequent to membrane binding, the C2A domain inserts residues on the Ca2+ binding loops into the membrane. Analysis of solution NMR measurements indicate that the domain inhabits two distinct structural states, with Ca2+ shifting the population between states towards a more rigid structure with greater affinity for PI(4,5)P2. Based on our results, we propose a mechanism where Ca2+ converts C2A from a structurally dynamic, low PI(4,5)P2 affinity state to a high affinity state that targets dysferlin to PI(4,5)P2 enriched membranes through interaction with Tyr23, K32, K33, and R77. Binding also involves changes in lipid packing and insertion by the third Ca2+ binding loop of the C2 domain into the membrane, which would contribute to dysferlin function in exocytosis and Ca2+ regulation.

Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding.

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.

Functional Role of an Unusual Transmembrane Acidic Residue in the Calcium Pump Regulator Myoregulin.

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 Ca2+-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 Ca2+ 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.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis.

Ca2+-dependent activator proteins for secretion (CAPSs) are required for Ca2+-regulated exocytosis in neurons and neuroendocrine cells. CAPSs contain a pleckstrin homology (PH) domain that binds PI(4,5)P2-membrane. There is also a C2 domain residing adjacent to the PH domain, but its function remains unclear. In this study, we solved the crystal structure of the CAPS-1 C2PH module. The structure showed that the C2 and PH tandem packs against one another mainly via hydrophobic residues. With this interaction, the C2PH module exhibited enhanced binding to PI(4,5)P2-membrane compared with the isolated PH domain. In addition, we identified a new PI(4,5)P2-binding site on the C2 domain. Disruption of either the tight interaction between the C2 and PH domains or the PI(4,5)P2-binding sites on both domains significantly impairs CAPS-1 function in Ca2+-regulated exocytosis at the Caenorhabditis elegans neuromuscular junction (NMJ). These results suggest that the C2 and PH domains constitute an effective unit to promote Ca2+-regulated exocytosis.

Hysteretic Pressure Dependence of Ca2+ Binding in LH1 Bacterial Membrane Chromoproteins.

Much of the thermodynamic parameter values that support life are set by the properties of proteins. While the denaturing effects of pressure and temperature on proteins are well documented, their precise structural nature is rarely revealed. This work investigates the destabilization of multiple Ca2+ binding sites in the cyclic LH1 light-harvesting membrane chromoprotein complexes from two Ca-containing sulfur purple bacteria by hydrostatic high-pressure perturbation spectroscopy. The native (Ca-saturated) and denatured (Ca-depleted) phases of these complexes are well distinguishable by much-shifted bacteriochlorophyll a exciton absorption bands serving as innate optical probes in this study. The pressure-induced denaturation of the complexes related to the failure of the protein Ca-binding pockets and the concomitant breakage of hydrogen bonds between the pigment chromophores and protein environment were found cooperative, involving all or most of the Ca2+ binding sites, but irreversible. The strong hysteresis observed in the spectral and kinetic characteristics of phase transitions along the compression and decompression pathways implies asymmetry in the relevant free energy landscapes and activation free energy distributions. A phase transition pressure equal to about 1.9 kbar was evaluated for the complexes from Thiorhodovibrio strain 970 from the pressure dependence of biphasic kinetics observed in the minutes to 100 h time range.

Crystal Structure Analysis of Sarcoplasmic-Calcium-Binding Protein: An Allergen in Scylla paramamosain.

The structure of allergenic proteins provides important information about the binding of allergens to antibodies. In this study, the crystal structure of Scy p 4 with a resolution of 1.60 Å was obtained by X-ray diffraction. Epitope mapping of Scy p 4 revealed that linear epitopes are located on the surface of Scy p 4. Also, conformational epitopes are mostly located in the structural conservative region. Further structural comparison, surface electrostatic potential, and hydrogen bond force analysis showed that mutation of Asp70 and Asp18/20/70 would lead to calcium-binding capacity being lost and destruction of allergenicity. Furthermore, a comparative analysis of structure showed that sarcoplasmic-calcium-binding protein (SCP) had high sequence, secondary, and spatial structural identity in crustaceans, which may be an important factor leading to cross-reactivity among crustaceans. The structure of Scy p 4 provides a template for epitope evaluation and localization of SCPs, which will help to reveal cross-reactivity among species.

Predictive Value of Combined Plasma D-Dimer, SCUBE1, and Right Ventricular Tei Index for the Prognosis of Elderly Patients with Acute Pulmonary Thromboembolism.

Acute pulmonary thromboembolism (APTE) has become a non-negligible clinical concern due to its high mortality and complex symptoms. Early diagnosis and prognostic assessment of APTE are of great significance for the long-term benefits of patients, especially elderly patients. Elderly patients with pulmonary embolism (n = 250) who presented to our hospital from January 2018 to July 2021 were recruited into this study. In addition, 50 healthy elderly people with no history of allergies were selected as the control group. An enzyme-linked immunosorbent assay (ELISA) method was used to determine concentrations of D-dimer and signal peptide-CUB-EGF domain-containing protein-1 (SCUBE1) in their plasma. Right ventricular volume contraction time (ICT), ejection time (ET), and isovolumic relaxation time (IRT) were determined by Doppler ultrasound. Right ventricular Tei index was calculated as (ICT + IRT)/ET. High plasma D-dimer, plasma SCUBE1, and right ventricular Tei index are risk factors for poor prognosis in APTE patients after treatment. Plasma D-dimer, plasma SCUBE1, and right ventricular Tei index have predictive value for poor prognosis in APTE patients. Their combined detection (0.256*DD +0.04*SCUBE1 + 10.188*Tei) can improve the sensitivity and specificity of prediction. There is a predictive value of combined plasma D-dimer, SCUBE1, and right ventricular Tei index for the prognosis of elderly patients with APTE.

Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin, dwarf open reading frame, endoregulin, phospholamban (PLB), and sarcolipin. We previously showed that these micropeptides assemble into homo-oligomeric complexes with varying affinity. Here, we tested whether different micropeptides can interact with each other, hypothesizing that coassembly into hetero-oligomers may affect micropeptide bioavailability to regulate SERCA. We quantified the relative binding affinity of each combination of candidates using automated fluorescence resonance energy transfer microscopy. All pairs were capable of interacting with good affinity, similar to the affinity of micropeptide self-binding (homo-oligomerization). Testing each pair at a 1:5 ratio and a reciprocal 5:1 ratio, we noted that the affinity of hetero-oligomerization of some micropeptides depended on whether they were the minority or majority species. In particular, sarcolipin was able to join oligomers when it was the minority species but did not readily accommodate other micropeptides in the reciprocal experiment when it was expressed in fivefold excess. The opposite was observed for endoregulin. PLB was a universal partner for all other micropeptides tested, forming avid hetero-oligomers whether it was the minority or majority species. Increasing expression of SERCA decreased PLB-dwarf open reading frame hetero-oligomerization, suggesting that SERCA-micropeptide interactions compete with micropeptide-micropeptide interactions. Thus, micropeptides populate a regulatory network of diverse protein assemblies. The data suggest that the complexity of this interactome increases exponentially with the number of micropeptides that are coexpressed in a particular tissue.

Construction of mouse cochlin mutants with different GAG-binding specificities and their use for immunohistochemistry.

Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.

Effects of membrane lipids on phospholamban pentameric channel structure and ion transportation mechanisms.

Protein-lipid interactions are an essential element of the function of many membrane ion-channel proteins. These potential interactions should be considered alongside the diversity and complexity of membrane lipid composition. Phospholamban (PLN) is an inhibitor of sarcoplasmic reticulum Ca2+ ATPase (SERCA). PLN is a 52-residue transmembrane protein encoded by lncRNA, and PLN monomers form stable pentamers of biological function in a lipid bilayer membrane. Some earlier studies suggest that it can form a cationic selective channel, while others suggest that it can only store ions. Here, we report the distribution of different lipids in the membrane and the structural dynamics and conductance properties of PLN pentamers after coarse-grained (CG) and all-atom (AA) molecular dynamics simulations of different systems. The results show that cholesterol is highly enriched around the protein and stabilizes the structure of the PLN pentamer. The absence of cholesterol increases the flexibility of the protein backbone. The conductance properties of monovalent ions and water suggest that they cannot spontaneously permeate through the PLN pentamer channel pore. However, the energy barrier to overcome is much lower in the absence of cholesterol, underlining the need to fully consider multiple lipid species when investigating small transmembrane protein oligomer ion-channel structure and conductance.

Structures of the TMC-1 complex illuminate mechanosensory transduction.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Chemical shift assignments of the C-terminal domain of CaBP1 bound to the IQ-motif of voltage-gated Ca2+ channel (CaV1.2).

The neuronal L-type voltage-gated Ca2+ channel (CaV1.2) interacts with Ca2+ binding protein 1 (CaBP1), that promotes Ca2+-induced channel activity. The binding of CaBP1 to the IQ-motif in CaV1.2 (residues 1644-1665) blocks the binding of calmodulin and prevents Ca2+-dependent inactivation of CaV1.2. This Ca2+-induced binding of CaBP1 to CaV1.2 is important for modulating neuronal synaptic plasticity, which may serve a role in learning and memory. Here we report NMR assignments of the C-terminal domain of CaBP1 (residues 99-167, called CaBP1C) that contains two Ca2+ bound at the third and fourth EF-hands (EF3 and EF4) and is bound to the CaV1.2 IQ-motif from CaV1.2 (BMRB accession no. 51518).

Conformational Change of the Hairpin-like-structured Robo2 Ectodomain Allows NELL1/2 Binding.

Since neural epidermal growth factor-like-like (NELL) 2 was identified as a novel ligand for the roundabout (Robo) 3 receptor, research on NELL-Robo signaling has become increasingly important. We have previously reported that Robo2 can bind to NELL1/2 in acidic conditions but not at neutral pH. The NELL1/2-binding site that is occluded in neutral conditions is thought to be exposed by a conformational change of the Robo2 ectodomain upon exposure to acidic pH; however, the underlying structural mechanisms are not well understood. Here, we investigated the interaction between the immunoglobulin-like domains and fibronectin type III domains that form hairpin-like structure of the Robo2 ectodomain, and demonstrated that acidic pH attenuates the interaction between them. Alternative splicing isoforms of Robo2, which affect the conformation of the hairpin-like structure, were found to have distinct NELL1/2-binding affinities. We developed Förster resonance energy transfer-based indicators for monitoring conformational change of the Robo2 ectodomain by individually inserting donor and acceptor fluorescent proteins at its ends. These experiments revealed that the ends of the Robo2 ectodomain are close to each other in acidic conditions. By combining these findings with the results of size exclusion chromatography analysis, we suggest that, in acidic conditions, the Robo2 ectodomain has a compact conformation with a loose hairpin-like structure. These results may help elucidate the signaling mechanisms resulting from the interaction between Robo2 and NELL1/2 in acidic conditions.

Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.

Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-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.