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.     

The C2A domain of double C2 protein gamma contains a functional nuclear localization signal

The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

Regulation of the mouse epithelial Ca2(+) channel TRPV6 by the Ca(2+)-sensor calmodulin

TRPV5 and TRPV6 are members of the superfamily of transient receptor potential (TRP) channels and facilitate Ca(2+) influx in a variety of epithelial cells. The activity of these Ca(2+) channels is tightly controlled by the intracellular Ca(2+) concentration in close vicinity to the channel mouth. The molecular mechanism underlying the Ca(2+)-dependent activity of TRPV5/TRPV6 is, however, still unknown. Here, the putative role of calmodulin (CaM) as the Ca(2+) sensor mediating the regulation of channel activity was investigated. Overexpression of Ca(2+)-insensitive CaM mutants (CaM(1234) and CaM(34)) significantly reduced the Ca(2+) as well as the Na(+) current of TRPV6- but not that of TRPV5-expressing HEK293 cells. By combining pull-down assays and co-immunoprecipitations, we demonstrated that CaM binds to both TRPV5 and TRPV6 in a Ca(2+)-dependent fashion. The binding of CaM to TRPV6 was localized to the transmembrane domain (TRPV6(327-577)) and consensus CaM-binding motifs located in the N (1-5-10 motif, TRPV6(88-97)) and C termini (1-8-14 motif, TRPV6(643-656)), suggesting a mechanism of regulation involving multiple interaction sites. Subsequently, chimeric TRPV6/TRPV5 proteins, in which the N and/or C termini of TRPV6 were substituted by that of TRPV5, were co-expressed with CaM(34) in HEK293 cells. Exchanging, the N and/or the C termini of TRPV6 by that of TRPV5 did not affect the CaM(34)-induced reduction of the Ca(2+) and Na(+) currents. These results suggest that CaM positively affects TRPV6 activity upon Ca(2+) binding to EF-hands 3 and 4, located in the high Ca(2+) affinity CaM C terminus, which involves the N and C termini and the transmembrane domain of TRPV6.     

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the μ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the μ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.     

The C2B domain of synaptotagmin I is a Ca2+-binding module

Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.     

The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.     

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.     

Characterization of microtubule binding domains in the Arabidopsis kinesin-like calmodulin binding protein.

The kinesin-like calmodulin binding protein (KCBP) is a new member of the kinesin superfamily that appears to be present only in plants. The KCBP is unique in its ability to interact with calmodulin in a Ca2+-dependent manner. To study the interaction of the KCBP with microtubules, we expressed different regions of the Arabidopsis KCBP and used the purified proteins in cosedimentation assays with microtubules. The motor domain with or without the calmodulin binding domain bound to microtubules. The binding of the motor domain containing the calmodulin binding region to microtubules was inhibited by Ca2+-calmodulin. This Ca2+-calmodulin regulation of motor domain interactions with microtubules was abolished in the presence of antibodies specific to the calmodulin binding region. In addition, the binding of the motor domain lacking the calmodulin binding region to microtubules was not inhibited in the presence of Ca2+-calmodulin, suggesting an essential role for the calmodulin binding region in Ca2+-calmodulin modulation. Results of the cosedimentation assays with the N-terminal tail suggest the presence of a second microtubule binding site on the KCBP. However, the interaction of the N-terminal tail region of the KCBP with microtubules was insensitive to ATP. These data on the interaction of the KCBP with microtubules provide new insights into the functioning of the KCBP in plants.     

Novel interaction of the voltage-dependent sodium channel (VDSC) with calmodulin: does VDSC acquire calmodulin-mediated Ca2+-sensitivity?

The voltage-dependent sodium channel (VDSC) interacts with intracellular molecules to modulate channel properties and localizations in neuronal cells. To study protein interactions, we applied yeast two-hybrid screening to the cytoplasmic C-terminal domain of the main pore-forming alpha-subunit. We found a novel interaction between the C-terminal domain and calmodulin (CaM). By two-hybrid interaction assays, we specified the interaction site of VDSC in a C-terminal region, which is composed of 38 amino acid residues and contains both IQ-like and Baa motifs. Using a fusion protein of the C-terminal domain, we showed that interaction with CaM occurred in the presence and absence of Ca(2+). Two synthetic peptides, each covering the IQ-like (NaIQ) or the Baa motifs (NaBaa), were used to examine the binding property by a gel mobility shift assay. Although the NaIQ and NaBaa sequences are overlapped, NaBaa binds only to Ca(2+)-bound Ca(2+)CaM, whereas NaIQ binds to both Ca(2+)CaM and Ca(2+)-free apoCaM. Fluorescence spectroscopy of dansylated CaM showed Ca(2+)-dependent spectral changes not only for NaBaa.CaM but also for NaIQ.CaM. The results, taken together with other results, indicate that whereas the NaBaa.CaM complex is formed in a Ca(2+)-dependent manner, the NaIQ.CaM complex has two conformational states, distinct with respect to the peptide binding site and the CaM conformation, depending on the Ca(2+) concentration. These observations suggest the possibility that VDSC is functionally modulated through the direct CaM interaction and the Ca(2+)-dependent conformational transition of the complex.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.     

Inositol hexakisphosphate (InsP6) can weaken the Ca(2+)-dependent membrane binding of C2AB domain of synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. In the present work, two fragments of the large cytoplasmic domain of synaptotagmin I, C2A and C2AB, were compared by combining surface plasmon resonance with circular dichroism and fluorescence techniques. C2AB and C2A had almost identical membrane binding constants, indicating that C2A is the predominate domain to bind to negatively charged phospholipids. After reacting with inositol hexakisphosphate (InsP6) a conformational change of C2AB was detected in the presence of liposome. The InsP6 binding notably weakened the Ca(2+)-dependent C2AB-membrane interaction, which suggests that InsP6 may act as a modulator of neurotransmitter release by altering the state of synaptotagmin-phospholipid interaction.     

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.     

The N-terminal domain of annexin 2 serves as a secondary binding site during membrane bridging

Annexin A2 (AnxA2) is a Ca(2+)- and acidic phospholipid-binding protein involved in many cellular processes. It undergoes Ca(2+)-mediated membrane bridging at neutral pH and has been demonstrated to be involved in an H(+)-mediated mechanism leading to a novel AnxA2-membrane complex structure. We used fluorescence techniques to characterize this H(+)-dependent mechanism at the molecular level; in particular, the involvement of the AnxA2 N-terminal domain. This domain was labeled at Cys-8 either with acrylodan or pyrene-maleimide fluorescent probes. Steady-state and time-resolved fluorescence analysis for acrylodan and fluorescence quenching by doxyl-labeled phospholipids revealed direct interaction between the N-terminal domain and the membrane. The absence of pyrene excimer suggested that interactions between N termini are not involved in the H(+)-mediated mechanism. These findings differ from those previously observed for the Ca(2+)-mediated mechanism. Protein titration experiments showed that the protein concentration for half-maximal membrane aggregation was twice for Ca(2+)-mediated compared with H(+)-mediated aggregation, suggesting that AnxA2 was able to bridge membranes either as a dimer or as a monomer, respectively. An N-terminally deleted AnxA2 was 2-3 times less efficient than the wild-type protein for H(+)-mediated membrane aggregation. We propose a model of AnxA2-membrane assemblies, highlighting the different roles of the N-terminal domain in the H(+)- and Ca(2+)-mediated membrane bridging mechanisms.     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

Structural determinants for Ca2+ and phosphatidylinositol 4,5-bisphosphate binding by the C2A domain of rabphilin-3A

Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca2+-dependent manner with a relatively high affinity (50 microm) in the presence of saturating concentrations of Ca2+. Moreover, IP3 and Ca2+ binding to the C2A domain mutually enhance each other. Here we present the Ca2+-bound solution structure of the C2A domain. Structural comparison with the previously published Ca2+-free crystal structure revealed that Ca2+ binding induces a conformational change of Ca2+ binding loop 3 (CBL3). Our IP3 binding studies as well as our IP3-C2A docking model show the active involvement of CBL3 in IP3 binding, suggesting that the conformational change on CBL3 upon Ca2+ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.     

Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity

Protein C is a vitamin K-dependent regulator of blood coagulation. It has beta-hydroxyaspartic acid in position 71 which is in the first of its two domains that are homologous to epidermal growth factor (EGF). This region has recently been demonstrated to have a Ca2+ binding site with a Kd of approximately 100 microM. Recombinant human protein C, expressed in mammalian tissue culture, had full biological activity and contained beta-hydroxyaspartic acid. Furthermore, it had a Ca2+-dependent epitope in the EGF-like domain, recognized by a monoclonal antibody. In contrast, a mutant recombinant human protein C in which beta-hydroxyaspartic acid had been replaced with glutamic acid in position 71 did not have the Ca2+-dependent epitope, and its biological activity was reduced to about 10% of normal. Fab' fragments of this antibody inhibited the anticoagulant activity of plasma-derived activated protein C, apparently by interfering with the interaction between activated protein C and its cofactor, protein S. The latter contains four tandemly arranged EGF homology domains. We propose that beta-hydroxyaspartic acid is directly involved in Ca2+ binding in protein C and in related proteins and that protein C interacts with protein S by means of its EGF homology regions.     

Association of L-type calcium channels with a vacuolar H(+)-ATPase G2 subunit

The class C L-type calcium (Ca(2+)) channels have been implicated in many important physiological processes. Here, we have identified a mouse vacuolar H(+)-ATPase (V-ATPase) G2 subunit protein that bound to the C-terminal domain of the pore-forming alpha(1C) subunit using a yeast two-hybrid screen. Protein-protein interaction between the V-ATPase G subunit and the alpha(1C) subunit was confirmed using in vitro GST pull-down assays and coimmunoprecipitation from intact cells. Moreover, treatment of cells expressing L-type Ca(2+) channels with a specific inhibitor of the V-ATPase blocked proper targeting of the channels to the plasma membrane.     

The C2A-C2B linker defines the high affinity Ca(2+) binding mode of rabphilin-3A

The Ca(2+) binding properties of C2 domains are essential for the function of their host proteins. We present here the first crystal structures showing an unexpected Ca(2+) binding mode of the C2B domain of rabphilin-3A in atomic detail. Acidic residues from the linker region between the C2A and C2B domains of rabphilin-3A interact with the Ca(2+)-binding region of the C2B domain. Because of these interactions, the coordination sphere of the two bound Ca(2+) ions is almost complete. Mutation of these acidic residues to alanine resulted in a 10-fold decrease in the intrinsic Ca(2+) binding affinity of the C2B domain. Using NMR spectroscopy, we show that this interaction occurred only in the Ca(2+)-bound state of the C2B domain. In addition, this Ca(2+) binding mode was maintained in the C2 domain tandem fragment. In NMR-based liposome binding assays, the linker was not released upon phospholipid binding. Therefore, this unprecedented Ca(2+) binding mode not only shows how a C2 domain increases its intrinsic Ca(2+) affinity, but also provides the structural base for an atypical protein-Ca(2+)-phospholipid binding mode of rabphilin-3A.     

Characterization of the Grp94/OS-9 Chaperone–Lectin Complex

Grp94 is a macromolecular chaperone belonging to the hsp90 family and is the most abundant glycoprotein in the endoplasmic reticulum (ER) of mammals. In addition to its essential role in protein folding, Grp94 was proposed to participate in the ER-associated degradation quality control pathway by interacting with the lectin OS-9, a sensor for terminally misfolded proteins. To understand how OS-9 interacts with ER chaperone proteins, we mapped its interaction with Grp94. Glycosylation of the full-length Grp94 protein was essential for OS-9 binding, although deletion of the Grp94 N-terminal domain relieved this requirement suggesting that the effect was allosteric rather than direct. Although yeast OS-9 is composed of a well-established N-terminal mannose recognition homology lectin domain and a C-terminal dimerization domain, we find that the C-terminal domain of OS-9 in higher eukaryotes contains “mammalian-specific insets” that are specifically recognized by the middle and C-terminal domains of Grp94. Additionally, the Grp94 binding domain in OS-9 was found to be intrinsically disordered. The biochemical analysis of the interacting regions provides insight into the manner by which the two associate and it additionally hints at a plausible biological role for the Grp94/OS-9 complex.