Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Novel aspects of calmodulin target recognition and activation.

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated proteins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca2+-pump, the Ca2+-activated K+-channel, the Ca2+/CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+-pump domain to only the C-terminal part of CaM (b) dimer formation with fragments of the K+-channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ ions to only one EF-hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM-dependent kinase IIalpha (CaMKIIalpha). In the latter complexes, CaM engulfs the CaM-binding domain peptide with its two Ca2+-binding lobes and forms a compact, ellipsoid-like complex. In the early 1990s, a model for the activation of CaM-regulated proteins was developed based on this observation and postulated activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes discussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM-regulated K+-channel and possibly also not for the Ca2+-pump.     

Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation

Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R(135)) and an 80 kDa isoform (4.1R(80)), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R(135) isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R(80) isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R(135) isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R(80). In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R(135) and 4.1R(80) bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R(80) was Ca2+-independent, whereas the binding of 4.1R(135) was strongly Ca2+-dependent. The HP of 4.1R(135) mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R(135) to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R(135) isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.     

Structural stabilization of protein 4.1R FERM domain upon binding to apo-calmodulin: novel insights into the biological significance of the calcium-independent binding of calmodulin to protein 4.1R

In erythrocytes, 4.1R80 (80 kDa isoform of protein 4.1R) binds to the cytoplasmic tail of the transmembrane proteins band 3 and GPC (glycophorin C), and to the membrane-associated protein p55 through the N- (N-terminal), α- (α-helix-rich) and C- (C-terminal) lobes of R30 [N-terminal 30 kDa FERM (4.1/ezrin/radixin/moesin) domain of protein 4.1R] respectively. We have shown previously that R30 binds to CaM (calmodulin) in a Ca2+-independent manner, the equilibrium dissociation constant (Kd) for R30-CaM binding being very similar (in the submicromolar range) in the presence or absence of Ca2+. In the present study, we investigated the consequences of CaM binding on R30's structural stability using resonant mirror detection and FTIR (Fourier-transform IR) spectroscopy. After a 30 min incubation above 40° C, R30 could no longer bind to band 3 or to GPC. In contrast, R30 binding to p55, which could be detected at a temperature as low as 34° C, was maintained up to 44° C in the presence of apo-CaM. Dynamic light scattering measurements indicated that R30, either alone or complexed with apo-CaM, did not aggregate up to 40° C. FTIR spectroscopy revealed that the dramatic variations in the structure of the β-sheet structure of R30 observed at various temperatures were minimized in the presence of apo-CaM. On the basis of Kd values calculated at various temperatures, ΔCp and ΔG° for R30 binding to apo-CaM were determined as -10 kJ · K(-1) · mol-1 and ~ -38 kJ · mol(-1) at 37° C (310.15 K) respectively. These data support the notion that apo-CaM stabilizes R30 through interaction with its β-strand-rich C-lobe and provide a novel function for CaM, i.e. structural stabilization of 4.1R80.     

Novel Mechanism of Regulation of Protein 4.1G Binding Properties Through Ca2+/Calmodulin-Mediated Structural Changes

Protein 4.1G (4.1G) is a widely expressed member of the protein 4.1 family of membrane skeletal proteins. We have previously reported that Ca²⁺-saturated calmodulin (Ca²⁺/CaM) modulates 4.1G interactions with transmembrane and membrane-associated proteins through binding to Four.one-ezrin–radixin–moesin (4.1G FERM) domain and N-terminal headpiece region (GHP). Here we identify a novel mechanism of Ca²⁺/CaM-mediated regulation of 4.1G interactions using a combination of small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy analyses. We document that GHP intrinsically disordered coiled structure switches to a stable compact structure upon binding of Ca²⁺/CaM. This dramatic conformational change of GHP inhibits in turn 4.1G FERM domain interactions due to steric hindrance. Based upon sequence homologies with the Ca²⁺/CaM-binding motif in protein 4.1R headpiece region, we establish that the 4.1G S⁷¹RGISRFIPPWLKKQKS peptide (pepG) mediates Ca²⁺/CaM binding. As observed for GHP, the random coiled structure of pepG changes to a relaxed globular shape upon complex formation with Ca²⁺/CaM. The resilient coiled structure of pepG, maintained even in the presence of trifluoroethanol, singles it out from any previously published CaM-binding peptide. Taken together, these results show that Ca²⁺/CaM binding to GHP, and more specifically to pepG, has profound effects on other functional domains of 4.1G.     

Structure of calmodulin bound to the hydrophobic IQ domain of the cardiac Ca(v)1.2 calcium channel

Ca2+-dependent inactivation (CDI) and facilitation (CDF) of the Ca(v)1.2 Ca2+ channel require calmodulin binding to a putative IQ motif in the carboxy-terminal tail of the pore-forming subunit. We present the 1.45 A crystal structure of Ca2+-calmodulin bound to a 21 residue peptide corresponding to the IQ domain of Ca(v)1.2. This structure shows that parallel binding of calmodulin to the IQ domain is governed by hydrophobic interactions. Mutations of residues I1672 and Q1673 in the peptide to alanines, which abolish CDI but not CDF in the channel, do not greatly alter the structure. Both lobes of Ca2+-saturated CaM bind to the IQ peptide but isoleucine 1672, thought to form an intramolecular interaction that drives CDI, is buried. These findings suggest that this structure could represent the conformation that calmodulin assumes in CDF.     

Decoding calcium signals by multifunctional CaM kinase.

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is one of the three major protein kinases coordinating cellular responses to hormones and neurotransmitters. It mediates the action of Ca2+ on neurotransmitter synthesis and release, on carbohydrate metabolism and on the cytoskeleton. CaM kinase has structural/functional properties that facilitate its response to distinctive attributes of Ca2+ signals which often involve transient increases that span a narrow concentration range and increases that are pulsatile rather than persistent. The kinase responds to the narrow working range of Ca2+ signals by the use of calmodulin as the Ca2+ sensor. It is activated by the binding of calmodulin to an autoinhibitory domain that keeps the kinase inactive in the basal state. The transient nature of the signal is accommodated by autophosphorylation of this autoinhibitory domain which allows the kinase to remain partially active after calmodulin dissociates and thereby switches it to a Ca(2+)-independent species. The pulsatile nature of Ca2+ signals may also be decoded by CaM kinase. Autophosphorylation traps calmodulin on autophosphorylated subunits by greatly reducing its off-rate. At high frequency of stimulation, calmodulin would remain trapped during the brief interval between Ca2+ oscillations and each successive rise in Ca2+ would recruit more calmodulin. This may enable a stimulus frequency dependent activation of CaM kinase.     

Calcium-dependent association of calmodulin with the C-terminal domain of the tetraspanin protein peripherin/rds

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 +/- 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function.     

The complex structure of calmodulin bound to a calcineurin peptide

The activity of the protein phosphatase calcineurin (CN) is regulated by an autoinhibition mechanism wherein several domains from its catalytic A subunit, including the calmodulin binding domain (CaMBD), block access to its active site. Upon binding of Ca2+ and calmodulin (Ca2+/CaM) to CaMBD, the autoinhibitory domains dissociate from the catalytic groove, thus activating the enzyme. To date, the structure of the CN/CaM/Ca2+ complex has not been determined in its entirety. Previously, we determined the structure of a fusion protein consisting of CaM and a 25-residue peptide taken from the CaMBD, joined by a 5-glycine linker. This structure revealed a novel CaM binding motif. However, the presence of the extraneous glycine linker cast doubt on the authenticity of this structure as an accurate representation of CN/CaM binding in vivo. Thus, here, we have determined the crystal structure of CaM complexed with the 25-residue CaMBD peptide without the glycine linker at a resolution of 2.1 A. The structure is essentially identical to the fusion construction which displays CaM bound to the CaMBD peptide as a dimer with an open, elongated conformation. The N-lobe from one molecule and C-lobe from another encompass and bind the CaMBD peptide. Thus, it validates the existence of this novel CaM binding motif. Our experiments suggest that the dimeric CaM/CaMBD complex exists in solution, which is unambiguously validated using a carefully-designed CaM-sepharose pull-down experiment. We discuss structural features that produce this novel binding motif, including the role of the CaMBD peptide residues Arg-408, Val-409, and Phe-410, which work to provide rigidity to the otherwise flexible central CaM helix joining the N- and C-lobes, ultimately keeping these lobes apart and forcing "head-to-tail" dimerization to attain the requisite N- and C-lobe pairing for CaMBD binding.     

Calmodulin and calmodulin binding proteins in amphibian rod outer segments

The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured. The molar ratio between rhodopsin and total CaM in ROS is 800:1. This is in good agreement with the data reported for bovine ROS CaM [Kohnken, R. E., Chafouleas, J. G., Eadie, D. M., Means, A. R., & McConnell, D.G. (1981) J. Biol. Chem. 256, 12517-12522]. In the absence of Ca2+, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca2+, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 X 10(-7) M Ca2+ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca2+-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca2+-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca2+. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. Moreover, while the Mr 140,000 band is not detected with DTSSP, a smeared band with a molecular weight between 78,000 and 93,000 is identified (with DTSSP) in the ROS membrane fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.     

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R⁸⁰ plays a key role in regulation of erythrocyte plasticity. Protein 4.1R⁸⁰ interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca²⁺-saturated calmodulin (Ca²⁺/CaM) through simultaneous binding to a short peptide (pep11; A²⁶⁴KKLWKVCVEHHTFFRL) and a serine residue (Ser¹⁸⁵), both located in the N-terminal 30 kDa FERM domain of 4.1R⁸⁰ (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca²⁺-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R⁸⁰, i.e. conservation of the pep11 sequence but substitution of the Ser¹⁸⁵ residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca²⁺-independent manner and that the Ca²⁺/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP₂) and other phospholipid species and that that PIP₂ inhibits Ca²⁺-free CaM but not Ca²⁺-saturated CaM binding to Cor30. We conclude that PIP₂ may play an important role as a modulator of apo-CaM binding to 4.1R⁸⁰ throughout evolution.     

Structural requirements for calmodulin binding to membrane-associated guanylate kinase homologs

Effector molecules such as calmodulin modulate the interactions of membrane-associated guanylate kinase homologs (MAGUKs) and other scaffolding proteins of the membrane cytoskeleton by binding to the Src homology 3 (SH3) domain, the guanylate kinase (GK) domain, or the connecting HOOK region of MAGUKs. Using surface plasmon resonance, we studied the interaction of members of all four MAGUK subfamilies--synapse-associated protein 97 (SAP97), calcium/calmodulin-dependent serine protein kinase (CASK), membrane palmitoylated protein 2 (MPP2), and zona occludens (ZO) 1--and calmodulin to determine interaction affinities and localize the binding site. The SH3-GK domains of the proteins and derivatives thereof were expressed in E. coli and purified. In all four proteins, high-affinity calmodulin binding was identified. CASK was shown to contain a Ca2+-dependent calmodulin binding site within the HOOK region, overlapping with a protein 4.1 binding site. In ZO1, a Ca2+-dependent calmodulin binding site was detected within the GK domain. The equilibrium dissociation constants for MAGUK-calmodulin interaction were found to range from 50 nM to 180 nM. Sequence analyses suggest that binding sites for calmodulin have evolved independently in at least three subfamilies. For ZO1, pulldown of GST-calmodulin was shown to occur in a calcium-dependent manner; moreover, molecular modeling and sequence analyses predict conserved basic residues to be exposed on one side of a helix. Thus, calmodulin binding appears to be a common feature of MAGUKs, and Ca2+-activated calmodulin may serve as a general regulator to affect the interactions of MAGUKs and various components of the cytoskeleton.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Interactions of calmodulin with metal ions and with its target proteins revealed by conformation-sensitive monoclonal antibodies

Two monoclonal antibodies (mAbs) raised against bovine calmodulin (CaM), CAM1 and CAM4, enable one to monitor conformational changes that occur in the molecule. The interaction of CAM1 with CaM depends on the Ca2+ occupancy of its Ca(2+)-binding sites. CAM4, in contrast, interacts with CaM in a Ca(2+)-independent manner, interacting with both holoCaM and EGTA-treated CaM to a similar extent. Their interaction with various CaMs, CaM tryptic fragments and chemically modified CaM, as well as molecular graphics, led to identification of the CAM1 and CAM4 epitopes on the C- and N-terminal lobes of CAM respectively. The two mAbs were used as macromolecular probes to detect conformational changes occurring in the CaM molecule upon binding of metal ions and target proteins and peptides. MAb CAM1 successfully detected changes associated with Al3+ binding even in the presence of Ca2+, indicating that Al3+ and Ca2+ ions may bind to the protein simultaneously, leading to a new conformation of the molecule. MAbs CAM1 and CAM4 were used to follow the interactions of CaM with its target peptides and proteins. Complexes with melittin, mastoparan, calcineurin and phosphodiesterase showed different immunological properties on an immuno-enzyme electrode, indicating unique structural properties for each complex.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling.

Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)

NHE1 (Na+/H+ exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K519R and R556FNKKYVKK, previously shown to mediate PIP2 (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (Kd=100-200?nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca2+/CaM (Ca2+-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na+ and Ca2+ concentrations.     

The solution structure of a plant calmodulin and the CaM-binding domain of the vacuolar calcium-ATPase BCA1 reveals a new binding and activation mechanism

The type IIb class of plant Ca(2+)-ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca(2+)-CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca(2+)-ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca(2+)-CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca(2+)-ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca(2+)-ATPases seems to occur in an organelle-specific manner.