The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

Characterization of Ca2+-ATPase, LMCA1, with native cell membrane nanoparticles system.

Ca2+-ATPases are membrane pumps that transport calcium ions across the cell membrane and are dependent on ATP. The mechanism of Listeria monocytogenes Ca2+-ATPase (LMCA1) in its native environment remains incompletely understood. LMCA1 has been investigated biochemically and biophysically with detergents in the past. This study characterizes LMCA1 using the detergent-free Native Cell Membrane Nanoparticles (NCMNP) system. As demonstrated by ATPase activity assays, the NCMNP7-25 polymer is compatible with a broad pH range and Ca2+ ions. This result suggests that NCMNP7-25 may have a wider array of applications in membrane protein research.

Crosstalk among Calcium ATPases: PMCA, SERCA and SPCA in Mental Diseases.

Calcium in mammalian neurons is essential for developmental processes, neurotransmitter release, apoptosis, and signal transduction. Incorrectly processed Ca2+ 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 Ca2+, 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 Ca2+-ATPase (PMCA), sarco/endoplasmic Ca2+-ATPase (SERCA) and secretory pathway Ca2+-ATPase (SPCA) are considered efficient line of defense against abnormal Ca2+ rises. However, their role is not limited only to Ca2+ 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 Ca2+-ATPases to neuropathology, with a special emphasis on mental diseases.

Identification of ATP2C1 mutations in the patients of Hailey-Hailey disease.

BACKGROUND: Familial benign chronic pemphigus, also known as Hailey-Hailey disease (HHD), is a clinically rare bullous Dermatosis. However the mechanism has not been clarified. The study aim to detect novel mutations in exons of ATP2C1 gene in HHD patients; to explore the possible mechnism of HHD pathogenesis by examining the expression profile of hSPCA1, miR-203, p63, Notch1 and HKII proteins in the skin lesions of HHD patients. METHODS: Genomic DNA was extracted from peripheral blood of HHD patients. All exons of ATP2C1 gene in HHD patients were amplified by PCR and the products were purified and sequenced. All related signaling proteins of interest were stained by using skin lesion tissues from HHD patients and miR-203 levels were also determined. RESULTS: One synonymous mutation c.G2598A (in exon 26), one nonsense mutation c.C635A and two missense mutations c.C1286A (p.A429D) and c. A1931G (p. D644G) were identified. The nonsense mutation changed codon UCG to stop codon UAG, causing a premature polypeptide chain of the functional region A. The two missense mutations were located in the region P (phosphorylation region) and the Mn binding site of hSPCA1. The level of hSPCA1 was significantly decreased in HHD patients compared to the normal human controls, accompanied by an increase of miR-203 level and a decrease of p63 and HKII levels. CONCLUSION: In our study, we found four mutations in HHD. Meanwhile we found increase of miR-203 level and a decrease of p63 and HKII levels. In addition, Notch1, which was negatively regulated p63, is downregulated. These factors may be involved in the signaling pathways of HHD pathogenesis. Our data showed that both p63 and miR-203 may have significant regulatory effects on Notch1 in the skin.

Physicochemical properties of tilapia (Oreochromis niloticus) actomyosin subjected to high intensity ultrasound in low NaCl concentrations.

Effects of high intensity ultrasound (HIU) on physicochemical properties of tilapia (Oreochromis niloticus) actomyosin in low NaCl concentrations were investigated. The protein content extracted in low NaCl concentrations (0.1-0.3 M NaCl) increased with increasing HIU intensity up to 20.62 W/cm2 (p < 0.05). The effect of HIU on actomyosin extractability in high NaCl concentrations (0.6 and 1.2 M NaCl) was less obvious. Ca2+-ATPase activity and total sulfhydryl (SH) group content decreased in both 0.2 and 0.6 M NaCl. HIU showed more pronounced effect on oxidation of the SH groups in 0.6 M NaCl, while the reactive SH content at 0.2 M NaCl increased after a prolonged exposure to HIU, suggesting conformational changes induced by HIU. Surface hydrophobicity of actomyosin in 0.6 M NaCl increased with increasing ultrasonic intensity and exposure time to a higher degree than that in 0.2 M NaCl. A greater absolute value of the zeta potential of actomyosin subjected to HIU were also observed. The HIU treatments decreased the turbidity of actomyosin incubated at 40 and 60 °C. A drastic increase in the solubility of myosin heavy chain (MHC) and actin with 0.2 M NaCl were evident when HIU treatments were applied, but degradation of MHC occurred in both 0.2 and 0.6 M NaCl. Based on particle size and microstructure, actomyosin in 0.6 M NaCl underwent more disruption by HIU than that in 0.2 M NaCl. HIU induced protein unfolding and protein dissociation, enabling better extraction in a lower NaCl concentration.

Autoinhibition and activation mechanisms of the eukaryotic lipid flippase Drs2p-Cdc50p.

The heterodimeric eukaryotic Drs2p-Cdc50p complex is a lipid flippase that maintains cell membrane asymmetry. The enzyme complex exists in an autoinhibited form in the absence of an activator and is specifically activated by phosphatidylinositol-4-phosphate (PI4P), although the underlying mechanisms have been unclear. Here we report the cryo-EM structures of intact Drs2p-Cdc50p isolated from S. cerevisiae in apo form and in the PI4P-activated form at 2.8 Å and 3.3 Å resolution, respectively. The structures reveal that the Drs2p C-terminus lines a long groove in the cytosolic regulatory region to inhibit the flippase activity. PIP4 binding in a cytosol-proximal membrane region triggers a 90° rotation of a cytosolic helix switch that is located just upstream of the inhibitory C-terminal peptide. The rotation of the helix switch dislodges the C-terminus from the regulatory region, activating the flippase.

Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic.

Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.

Structure and autoregulation of a P4-ATPase lipid flippase.

Type 4 P-type ATPases (P4-ATPases) are lipid flippases that drive the active transport of phospholipids from exoplasmic or luminal leaflets to cytosolic leaflets of eukaryotic membranes. The molecular architecture of P4-ATPases and the mechanism through which they recognize and transport lipids have remained unknown. Here we describe the cryo-electron microscopy structure of the P4-ATPase Drs2p-Cdc50p, a Saccharomyces cerevisiae lipid flippase that is specific to phosphatidylserine and phosphatidylethanolamine. Drs2p-Cdc50p is autoinhibited by the C-terminal tail of Drs2p, and activated by the lipid phosphatidylinositol-4-phosphate (PtdIns4P or PI4P). We present three structures that represent the complex in an autoinhibited, an intermediate and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of autoinhibition and PI4P-dependent activation. We also observe a putative lipid translocation pathway in this flippase that involves a conserved PISL motif in transmembrane segment 4 and polar residues of transmembrane segments 2 and 5, in particular Lys1018, in the centre of the lipid bilayer.

An N-terminal Ca2+-binding motif regulates the secretory pathway Ca2+/Mn2+-transport ATPase SPCA1.

The Ca2+/Mn2+ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and are implicated in breast cancer and Hailey-Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. We observed that the purified SPCA1a displays a lower Ca2+ affinity and slightly lower Mn2+ affinity than SPCA2. Remarkably, the turnover rates of SPCA1a in the presence of Mn2+ and SPCA2 incubated with Ca2+ and Mn2+ were comparable, whereas the turnover rate of SPCA1a in Ca2+ was 2-fold higher. Moreover, we noted an unusual biphasic activation curve for the SPCA1a ATPase and autophosphorylation activity, not observed with SPCA2. We also found that the biphasic pattern and low apparent ion affinity of SPCA1a critically depends on ATP concentration. We further show that the specific properties of SPCA1a at least partially depend on an N-terminal EF-hand-like motif, which is present only in the SPCA1a isoform and absent in SPCA2. This motif binds Ca2+, and its mutation lowered the Ca2+ turnover rate relative to that of Mn2+, increased substrate affinity, and reduced the level of biphasic activation of SPCA1a. A biochemical analysis indicated that Ca2+ binding to the N-terminal EF-hand-like motif promotes the activity of SPCA1a by facilitating autophosphorylation. We propose that this regulation may be physiologically relevant in cells with a high Ca2+ load, such as mammary gland cells during lactation, or in cells with a low ATP content, such as keratinocytes.

The PQ-loop protein Any1 segregates Drs2 and Neo1 functions required for viability and plasma membrane phospholipid asymmetry.

Membrane asymmetry is a key organizational feature of the plasma membrane. Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that establish membrane asymmetry by translocating phospholipids, such as phosphatidylserine (PS) and phospatidylethanolamine, from the exofacial leaflet to the cytosolic leaflet. Saccharomyces cerevisiae expresses five P4-ATPases: Drs2, Neo1, Dnf1, Dnf2, and Dnf3. The inactivation of Neo1 is lethal, suggesting Neo1 mediates an essential function not exerted by the other P4-ATPases. However, the disruption of ANY1, which encodes a PQ-loop membrane protein, allows the growth of neo1Δ and reveals functional redundancy between Golgi-localized Neo1 and Drs2. Here we show Drs2 PS flippase activity is required to support neo1Δ any1Δ viability. Additionally, a Dnf1 variant with enhanced PS flipping ability can replace Drs2 and Neo1 function in any1Δ cells. any1Δ also suppresses drs2Δ growth defects but not the loss of membrane asymmetry. Any1 overexpression perturbs the growth of cells but does not disrupt membrane asymmetry. Any1 coimmunoprecipitates with Neo1, an association prevented by the Any1-inactivating mutation D84G. These results indicate a critical role for PS flippase activity in Golgi membranes to sustain viability and suggests Any1 regulates Golgi membrane remodeling through protein-protein interactions rather than a previously proposed scramblase activity.

Acceleration of cryo-EM Flexible Fitting for Large Biomolecular Systems by Efficient Space Partitioning.

Flexible fitting is a powerful technique to build the 3D structures of biomolecules from cryoelectron microscopy (cryo-EM) density maps. One popular method is a cross-correlation coefficient-based approach, where the molecular dynamics (MD) simulation is carried out with the biasing potential that includes the cross-correlation coefficient between the experimental and simulated density maps. Here, we propose efficient parallelization schemes for the calculation of the cross-correlation coefficient to accelerate flexible fitting. Our schemes are tested for small, medium, and large biomolecules using CPU and hybrid CPU + GPU architectures. The scheme for the atomic decomposition MD is suitable for small proteins such as Ca2+-ATPase with the all-atom Go model, while that for the domain decomposition MD is better for larger systems such as ribosome with the all-atom Go or the all-atom explicit solvent models. Our methods allow flexible fitting for various biomolecules with reasonable computational cost. This approach also connects high-resolution structure refinements with investigation of protein structure-function relationship.

E2P-like states of plasma membrane Ca2+­ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

The plasma membrane Ca2+­ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+­ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+­ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

SERCA plays a crucial role in the toxicity of a betulinic acid derivative with potential antimalarial activity.

Malaria is one of the most significant infectious diseases that affect poor populations in tropical areas throughout the world. Plants have been shown to be a good source for the development of new antimalarial chemotherapeutic agents, as shown for the discovery of quinine and artemisinin derivatives. Our research group has been working with semisynthetic triterpene derivatives that show potential antimalarial activity toward different strains of Plasmodium falciparum by specifically modulating calcium pathways in the parasite. Promising results were obtained for nanomolar concentrations of the semisynthetic betulinic acid derivative LAFIS13 against the P. falciparum 3D7 strain in vitro, with a selectivity index of 18 compared to a mammalian cell line. Continuing these studies, we present here in vitro and in vivo toxicological evaluations of this compound, followed by docking studies with PfATP6, a sarco/endoplasmic reticulum Ca+2-ATPase (SERCA) protein. LAFIS13 showed an LD50 between 300 and 50 mg/kg, and the acute administration of 50 mg/kg (i.p.) had no negative effects on hematological, biochemical and histopathological parameters. Based on the results of the in vitro assays, LAFIS13 not exerted significant effects on coagulation parameters of human peripheral blood, but a hemolytic activity was verified at higher concentrations. According to the molecular docking study, the PfATP6 protein may be a target for LAFIS13, which corroborates its previously reported modulatory effects on calcium homeostasis in the parasite. Notably, LAFIS13 showed a higher selectivity for the mammalian SERCA protein than for PfATP6, thus impairing the selectivity between parasite and host. In summary, the direct interaction with calcium pumps and the hemolytic potential of the compound proved to be plausible mechanism of LAFIS13 toxicity.

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni.

Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a duplication event. At the protein level, SMA1 and SMA2 only slightly differ in length and in the sequence of the nucleotide-binding domain. To get functional information on SMA1, we produced it in an active form in Saccharomyces cerevisiae, as previously done for SMA2. Using phosphorylation assays from ATP, we demonstrated that like SMA2, SMA1 bound calcium in a cooperative mode with an apparent affinity in the micromolar range. We also showed that SMA1 and SMA2 had close sensitivities to cyclopiazonic acid but different sensitivities to thapsigargin, two specific inhibitors of SERCA pumps. On the basis of transcriptomic data available in GeneDB, we hypothesize that SMA1 is a housekeeping Ca2+-ATPase, whereas SMA2 might be required in particular striated-like muscles like those present the tail of the cercariae, the infecting form of the parasite.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Protein-phospholipid interplay revealed with crystals of a calcium pump.

The lipid bilayer has so far eluded visualization by conventional crystallographic methods, severely limiting our understanding of phospholipid- and protein-phospholipid interactions. Here we describe electron density maps for crystals of Ca2+-ATPase in four different states obtained by X-ray solvent contrast modulation. These maps resolve the entire first layer of phospholipids surrounding the transmembrane helices, although less than half of them are hydrogen-bonded to protein residues. Phospholipids follow the movements of associated residues, causing local distortions and changes in thickness of the bilayer. Unexpectedly, the entire protein tilts during the reaction cycle, governed primarily by a belt of Trp residues, to minimize energy costs accompanying the large perpendicular movements of the transmembrane helices. A class of Arg residues extend their side chains through the cytoplasm to exploit phospholipids as anchors for conformational switching. Thus, phospholipid-Arg/Lys and phospholipid-Trp interactions have distinct functional roles in the dynamics of ion pumps and, presumably, membrane proteins in general.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

The Ca2+-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

Ca2+ transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca2+ homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca2+ uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca2+ and other ions across the SR. During Ca2+ uptake by the SR Ca2+-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca2+ transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca2+ release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca2+ transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

Evidence of the presence of a calmodulin-sensitive plasma membrane Ca2+-ATPase in Trypanosoma equiperdum.

Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca2+ homeostasis. Therefore, the plasma membrane Ca2+-ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif.

Nucleotide Binding in an Engineered Recombinant Ca2+-ATPase N-Domain.

A recombinant Ca2+-ATPase nucleotide binding domain (N-domain) harboring the mutations Trp552Leu and Tyr587Trp was expressed and purified. Chemical modification by N-bromosuccinimide and fluorescence quenching by acrylamide showed that the displaced Trp residue was located at the N-domain surface and slightly exposed to solvent. Guanidine hydrochloride-mediated N-domain unfolding showed the low structural stability of the α6-loop-α7 motif (the new Trp location) located near the nucleotide binding site. The binding of nucleotides (free and in complex with Mg2+) to the engineered N-domain led to significant intrinsic fluorescence quenching (ΔFmax ∼ 30%) displaying a saturable hyperbolic pattern; the calculated affinities decreased in the following order: ATP > ADP = ADP-Mg2+ > ATP-Mg2+. Interestingly, it was found that Ca2+ binds to the N-domain as monitored by intrinsic fluorescence quenching (ΔFmax ∼ 12%) with a dissociation constant (Kd) of 50 µM. Notably, the presence of Ca2+ (200 µM) increased the ATP and ADP affinity but favored the binding of ATP over that of ADP. In addition, binding of ATP to the N-domain generated slight changes in secondary structure as evidenced by circular dichroism spectral changes. Molecular docking of ATP to the N-domain provided different binding modes that potentially might be the binding stages prior to γ-phosphate transfer. Finally, the nucleotide binding site was studied by fluorescein isothiocyanate labeling and molecular docking. The N-domain of Ca2+-ATPase performs structural dynamics upon Ca2+ and nucleotide binding. It is proposed that the increased affinity of the N-domain for ATP mediated by Ca2+ binding may be involved in Ca2+-ATPase activation under normal physiological conditions.