[Show abstract][Hide abstract] ABSTRACT: The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca2+ -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor
residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal
lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G.,
Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015–4024). This is a relatively rapid reaction, with an apparent half-time of ∼1 s. The dissociation of CaM from PMCA4b
or C28 is much slower, with an overall half-time of ∼10 min. Using targeted molecular dynamics, we now show that dissociation
of Ca2+-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM,
leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110,
removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics
calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from
that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA
primed for activation.
[Show abstract][Hide abstract] ABSTRACT: The "w" splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.
Full-text · Article · Jun 2011 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Kinetic studies of biochemical reactions are typically carried out in a dilute solution that rarely contains anything more than reactants, products, and buffers. In such studies, mass-action-based kinetic models are used to analyze the progress curves. However, intracellular compartments are crowded by macromolecules. Therefore, we investigated the adequacy of the proposed generalizations of the mass-action model, which are meant to describe reactions in crowded media. To validate these models, we measured time-resolved kinetics for dansylamide binding to carbonic anhydrase in solutions crowded with polyethylene glycol and Ficoll. The measured progress curves clearly show the effects of crowding. The fractal-like model proposed by Savageau was used to fit these curves. In this model, the association rate coefficient k(a) allometrically depends on concentrations of reactants. We also considered the fractal kinetic model proposed by Schnell and Turner, in which k(a) depends on time according to a Zipf-Mandelbrot distribution, and some generalizations of these models. We found that the generalization of the mass-action model, in which association and dissociation rate coefficients are concentration-dependent, represents the preferred model. Other models based on time-dependent rate coefficients were inadequate or not preferred by model selection criteria.
Full-text · Article · May 2011 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: Although membrane proteins constitute more than 20% of the total proteins, the structures of only a few are known in detail. An important group of integral membrane proteins are ion-transporting ATPases of the P-type family, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. There are several crystal structures of the sarcoplasmic reticulum Ca(2+) pump (SERCA) revealing different conformations, and recently, crystal structures of the H(+)-ATPase and the Na(+)/K(+)-ATPase were reported as well. However, there are no atomic resolution structures for other P-type ATPases including the plasma membrane calcium pump (PMCA), which is integral to cellular Ca(2+) signaling. Crystallization of these proteins is challenging because there is often no natural source from which the protein can be obtained in large quantities, and the presence of multiple isoforms in the same tissue further complicates efforts to obtain homogeneous samples suitable for crystallization. Alternative techniques to study structural aspects and conformational transitions in the PMCAs (and other P-type ATPases) have therefore been developed. Specifically, information about the structure and assembly of the transmembrane domain of an integral membrane protein can be obtained from an analysis of the lipid-protein interactions. Here, we review recent efforts using different hydrophobic photo-labeling methods to study the non-covalent interactions between the PMCA and surrounding phospholipids under different experimental conditions, and discuss how the use of these lipid probes can reveal valuable information on the membrane organization and conformational state transitions in the PMCA, Na(+)/K(+)-ATPase, and other P-type ATPases.
Full-text · Article · May 2011 · Current Chemical Biology
[Show abstract][Hide abstract] ABSTRACT: The exposure of plasma membrane calcium pump (PMCA) to surrounding phospholipids was assessed by measuring the incorporation
of the photoactivatable phosphatidylcholine analogue [125I]TID-PC/16 to the protein. In the presence of Ca2+ both calmodulin
(CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with
V8 protease results in 3 main fragments: N which includes transmembrane segments M1 and M2, M which includes M3 and M4 and
C which includes M5 to M10.
CaM decreased the level of incorporation of [125I]TID-PC/16 to fragments M and C, while phosphatidic acid decreased the incorporation
of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend
to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations
produced by CaM and PA. To verify this point, we measured RET between PMCA labeled with eosin-isothiocyanate at the ATP-binding
site and the phospholipid Rho-PE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these
two probes while PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site
and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced
by CaM or PA and show that those differences involve transmembrane regions.
Full-text · Article · Mar 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation
of the photoactivatable phosphatidylcholine analog [125I]TID-PC/16 to the protein. In the presence of Ca2+ both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [125I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane
segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation
of [125I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [125I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to
the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced
by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate
at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer
between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between
the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between
PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.
Full-text · Article · Mar 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Ca2+ and Ca2+-dependent signals are essential for sperm maturation and fertilization. In mouse sperm the plasma membrane Ca2+-ATPase (PMCA) isoform 4 plays a crucial role in Ca2+ transport. The two major splice variants of PMCA4 are PMCA4a and PMCA4b. PMCA4a differs from PMCA4b in the mechanism of calmodulin
binding and activation. PMCA4a shows a much higher basal activity and is more effective than PMCA4b in returning Ca2+ to resting levels. Knock-out mice carrying a PMCA4-null mutation are infertile because their sperm cannot achieve a hyperactivated
state of motility. As sperm reach functional maturity during their transit through the epididymis, the expression of PMCA4a
and 4b was assessed in bull testis and epididymis. Quantitative PCR revealed that PMCA4b is the major splice variant in testis,
caput, and corpus epididymidis. In contrast, PMCA4a is the major splice variant in cauda epididymidis, whereas sperm are transcriptionally
silent. Immunohistochemical staining using a new antibody against bovine PMCA4a located the PMCA4a to the apical membrane
of the epithelium of cauda epididymidis, whereas testis, caput, and corpus epididymidis were negative. Western blotting of
testis, epididymis, and sperm isolated from caput and cauda epididymidis showed a much higher level of PMCA4a in cauda epididymidis
and sperm from cauda epididymidis compared with testis membranes and sperm from caput epididymidis. These findings suggest
that PMCA4a is transferred to bovine sperm membranes in cauda epididymidis. This isoform switch may facilitate a higher calcium
turnover in sperm necessary to traverse the female genital tract.
Full-text · Article · Dec 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Myo10 is an unconventional myosin with important functions in filopodial motility, cell migration, and cell adhesion. The neck region of Myo10 contains three IQ motifs that bind calmodulin (CaM) or the tissue-restricted calmodulin-like protein (CLP) as light chains. However, little is known about the mechanism of light chain binding to the IQ motifs in Myo10. Binding of CaM and CLP to each IQ motif was assessed by nondenaturing gel electrophoresis and by stopped-flow experiments using fluorescence-labeled CaM and CLP. Although the binding kinetics are different in each case, there are similarities in the mechanism of binding of CaM and CLP to IQ1 and IQ2: for both IQ motifs Ca(2+) increased the binding affinity, mainly by increasing the rate of the forward steps. The general kinetic mechanism comprises a two-step process, which in some cases may involve the binding of a second IQ motif with lower affinity. For IQ3, however, the kinetics of CaM binding is very different from that of CLP. In both cases, binding in the absence of Ca(2+) is poor, and addition of Ca(2+) decreases the K(d) to below 10 nM. However, while the CaM binding kinetics are complex and best fitted by a multistep model, binding of CLP is fitted by a relatively simple two-step model. The results show that, in keeping with growing structural evidence, complexes between CaM or CaM-like myosin light chains and IQ motifs are highly diverse and depend on the specific sequence of the particular IQ motif as well as the light chain.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca(2+) pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2'-[(125)I]iodo-4'-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([(125)I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [(125)I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca(2+). This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca(2+) and calmodulin decreases by approximately the same amount. However, incubation with Ca(2+) alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [(125)I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E(1) conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca(2+) + C28 for SERCA and Ca(2+) alone for PMCA), and one in which the enzyme is fully active (Ca(2+) for SERCA and Ca(2+)-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.
Full-text · Article · Feb 2009 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.
[Show abstract][Hide abstract] ABSTRACT: Calmodulin-like protein (CLP) is a specific light chain of unconventional myosin-10 (Myo10) and enhances Myo10-dependent filopodial extension. Here we show that phenylalanine-795 in the third IQ domain (IQ3) of Myo10 is critical for CLP binding. Remarkably, mutation of F795 to alanine had little effect on calmodulin binding to IQ3. Fluorescence microscopy and time-lapse video microscopy showed that HeLa cells expressing CLP and transiently transfected with GFP-Myo10-F795A exhibited significantly shorter filopodia and decreased intrafilopodial motility compared to wildtype GFP-Myo10-transfected cells. Thus, F795 represents a unique anchor for CLP and is essential for CLP-mediated Myo10 function in filopodial extension and motility.
[Show abstract][Hide abstract] ABSTRACT: Plasma-membrane calcium pumps [PMCAs (plasma-membrane Ca(2+)-ATPases)] expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. Recent work indicates functional versatility among PMCA isoforms, with specific pumps being essential for cochlear hair cell function, sperm motility, feedback signalling in the heart and pre- and post-synaptic Ca(2+) regulation in neurons. The functional versatility of PMCAs is due to differences in their regulation by CaM (calmodulin), kinases and other signalling proteins, as well as to their differential targeting and retention in defined plasma membrane domains. The basis for this is the structural diversity of PMCAs. In mammals, four genes encode PMCA isoforms 1-4, and each of these has multiple variants generated by alternative RNA splicing. The alternatively spliced regions are intimately involved in the regulatory interactions and differential membrane localization of the pumps. The alternatively spliced C-terminal tail acts as an autoinhibitory domain by interacting with the catalytic core of the pump. The degree of inhibition and the kinetics of interaction with the major activator CaM differ between PMCA variants. This translates into functional differences in how PMCAs handle Ca(2+) signals of different magnitude and frequency. Accumulating evidence thus demonstrates how structural diversity provides functional versatility in the PMCAs.
Full-text · Article · Dec 2007 · Biochemical Society Transactions
[Show abstract][Hide abstract] ABSTRACT: The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump
(PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those
of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates
of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow
experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a,
an “open” conformation with high activity, and a “closed” one with lower activity. Compared with PMCA4b (Penheiter, A. R.,
Bajzer, Z., Filoteo, A. G., Thorogate, R., Török, K., and Caride, A. J. (2003) Biochemistry 41, 12115–12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between
the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations
in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev,
N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500–24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties
of PMCA isoforms.