Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.     

Insights into modulation of calcium signaling by magnesium in calmodulin, troponin C and related EF-hand proteins

The Ca²⁺-binding helix-loop-helix structural motif called “EF-hand” is a common building block of a large family of proteins that function as intracellular Ca²⁺-receptors. These proteins respond specifically to micromolar concentrations of Ca²⁺ in the presence of ~1000-fold excess of the chemically similar divalent cation Mg²⁺. The intracellular free Mg²⁺ concentration is tightly controlled in a narrow range of 0.5-1.0 mM, which at the resting Ca²⁺ levels is sufficient to fully or partially saturate the Ca²⁺-binding sites of many EF-hand proteins. Thus, to convey Ca²⁺ signals, EF-hand proteins must respond differently to Ca²⁺ than to Mg²⁺. In this review the structural aspects of Mg²⁺ binding to EF-hand proteins are considered and interpreted in light of the recently proposed two-step Ca²⁺-binding mechanism (Grabarek, Z., J. Mol. Biol., 2005, 346, 1351). It is proposed that, due to stereochemical constraints imposed by the two-EF-hand domain structure, the smaller Mg²⁺ ion cannot engage the ligands of an EF-hand in the same way as Ca²⁺ and defaults to stabilizing the apo-like conformation of the EF-hand. It is proposed that Mg²⁺ plays an active role in the Ca²⁺-dependent regulation of cellular processes by stabilizing the “off state” of some EF-hand proteins, thereby facilitating switching off their respective target enzymes at the resting Ca²⁺ levels. Therefore, some pathological conditions attributed to Mg²⁺ deficiency might be related to excessive activation of underlying Ca²⁺-regulated cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Effects of Metal-Binding Properties of Human Kv Channel-Interacting Proteins on their Molecular Structure and Binding with Kv4.2 Channel

The goal of the present study is to explore whether Ca²⁺ and Mg²⁺-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca²⁺-binding sites, respectively, and only one type of Mg²⁺-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca²⁺, but the binding affinity for Mg²⁺ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg²⁺ and Ca²⁺. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg²⁺ and Ca²⁺. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg²⁺- and Ca²⁺-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca²⁺-bound form in the dark to a Mg²⁺-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca²⁺. Substitutions affect residues directly involved in Ca²⁺ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca²⁺ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca²⁺ and blocking the enzyme in a constitutively active state at physiological levels of Ca²⁺. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca²⁺-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.     

The renaissance of Ca2+-binding proteins in the nervous system: secretagogin takes center stage

Effective control of the Ca²⁺ homeostasis in any living cell is paramount to coordinate some of the most essential physiological processes, including cell division, morphological differentiation, and intercellular communication. Therefore, effective homeostatic mechanisms have evolved to maintain the intracellular Ca²⁺ concentration at physiologically adequate levels, as well as to regulate the spatial and temporal dynamics of Ca²⁺signaling at subcellular resolution. Members of the superfamily of EF-hand Ca²⁺-binding proteins are effective to either attenuate intracellular Ca²⁺ transients as stochiometric buffers or function as Ca²⁺ sensors whose conformational change upon Ca²⁺ binding triggers protein-protein interactions, leading to cell state-specific intracellular signaling events. In the central nervous system, some EF-hand Ca²⁺-binding proteins are restricted to specific subtypes of neurons or glia, with their expression under developmental and/or metabolic control. Therefore, Ca²⁺-binding proteins are widely used as molecular markers of cell identity whilst also predicting excitability and neurotransmitter release profiles in response to electrical stimuli. Secretagogin is a novel member of the group of EF-hand Ca²⁺-binding proteins whose expression precedes that of many other Ca²⁺-binding proteins in postmitotic, migratory neurons in the embryonic nervous system. Secretagogin expression persists during neurogenesis in the adult brain, yet becomes confined to regionalized subsets of differentiated neurons in the adult central and peripheral nervous and neuroendocrine systems. Secretagogin may be implicated in the control of neuronal turnover and differentiation, particularly since it is re-expressed in neoplastic brain and endocrine tumors and modulates cell proliferation in vitro. Alternatively, and since secretagogin can bind to SNARE proteins, it might function as a Ca²⁺ sensor/coincidence detector modulating vesicular exocytosis of neurotransmitters, neuropeptides or hormones. Thus, secretagogin emerges as a functionally multifaceted Ca²⁺-binding protein whose molecular characterization can unravel a new and fundamental dimension of Ca²⁺signaling under physiological and disease conditions in the nervous system and beyond.     

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca²⁺-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca²⁺-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca²⁺ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca²⁺-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca²⁺ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca²⁺ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca²⁺-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca²⁺ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca²⁺-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca²⁺-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca²⁺-binding proteins.     

Ca<Superscript>2+</Superscript> binding protein-1 inhibits Ca<Superscript>2+</Superscript> currents and exocytosis in bovine chromaffin cells

Summary Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca²⁺ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na⁺ and Ca²⁺ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca²⁺] _i response evoked by high K⁺ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca²⁺ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect Na⁺, Ca²⁺ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca²⁺] _i responses elicited by high-K⁺ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca²⁺ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells. M.-L. Chen and Y.-C. Chen contributed equally to this study     

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery

EF-hand Ca²⁺-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca²⁺. Upon binding Ca²⁺, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca²⁺-binding affinity of CaM and most S100s in the absence of target is weak (^CaK_D > 1 μM). However, upon effector protein binding, the Ca²⁺ affinity of these proteins increases via heterotropic allostery (^CaK_D < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca²⁺ homeostasis and (ii) strict maintenance of Ca²⁺-signaling within a narrow dynamic range of free Ca²⁺ ion concentrations, [Ca²⁺]^free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca²⁺ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.     

Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca2+ Binding Affinity

The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca²⁺ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca²⁺-binding loops and non Ca²⁺-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca²⁺-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca²⁺-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca²⁺-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca²⁺-binding affinity constants of EF-hand proteins. The web server is freely available at http://202.41.10.46/calb/index.html.     

Plasma membrane calcium ATPase isoform 3 expression in single cells isolated from rat liver

Photon absorption by photoreceptors activates hydrolysis of cGMP, which shuts down cGMP-gated channels and decreases free Ca²⁺ concentrations in outer segment. Suppression of Ca²⁺ influx through the cGMP channel by light activates retinal guanylyl cyclase through guanylyl cyclase activating proteins (GCAPs) and thus expedites photoreceptors recovery from excitation and restores their light sensitivity. GCAP1 and GCAP2, two ubiquitous among vertebrate species isoforms of GCAPs that activate retGC during rod response to light, are myristoylated Ca²⁺/Mg²⁺-binding proteins of the EF-hand superfamily. They consist of one non-metal binding EF-hand-like domain and three other EF-hands, each capable of binding Ca²⁺ and Mg²⁺. In the metal binding EF-hands of GCAP1, different point mutations can selectively block binding of Ca²⁺ or both Ca²⁺ and Mg²⁺ altogether. Activation of retGC at low Ca²⁺ (light adaptation) or its inhibition at high Ca²⁺ (dark adaptation) follows a cycle of Ca²⁺/Mg²⁺ exchange in GCAPs, rather than release of Ca²⁺ and its binding by apo-GCAPs. The Mg²⁺ binding in two of the EF-hands controls docking of GCAP1 with retGC1 in the conditions of light adaptation and is essential for activation of retGC. Mg²⁺ binding in a C-terminal EF-hand contributes to neither retGC1 docking with the cyclase nor its subsequent activation in the light, but is specifically required for switching the cyclase off in the conditions of dark adaptation by binding Ca²⁺. The Mg²⁺/Ca²⁺ exchange in GCAP1 and 2 operates within different range of intracellular Ca²⁺ concentrations and provides a two-step activation of the cyclase during rod recovery.     

The Potassium Channel Interacting Protein 3 (DREAM/KChIP3) Heterodimerizes with and Regulates Calmodulin Function

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca²⁺) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca²⁺, DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca²⁺. In the absence of Ca²⁺, DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca²⁺-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 μm as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional ¹H,¹⁵N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to ¹⁵N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca²⁺-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca²⁺ also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH₂-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.     

Physiologically Relevant Free Ca2+ Ion Concentrations Regulate STRA6-Calmodulin Complex Formation via the BP2 Region of STRA6

The interaction of calmodulin (CaM) with the receptor for retinol uptake, STRA6, involves an α-helix termed BP2 that is located on the intracellular side of this homodimeric transporter (Chen et al., 2016 [1]). In the absence of Ca²⁺, NMR data showed that a peptide derived from BP2 bound to the C-terminal lobe (C-lobe) of Mg²⁺-bound CaM (^MgCaM). Upon titration of Ca²⁺ into ^MgCaM-BP2, NMR chemical shift perturbations (CSPs) were observed for residues in the C-lobe, including those in the EF-hand Ca²⁺-binding domains, EF3 and EF4 (^CaK_D = 60 ± 7 nM). As higher concentrations of free Ca²⁺ were achieved, CSPs occurred for residues in the N-terminal lobe (N-lobe) including those in EF1 and EF2 (^CaK_D = 1000 ± 160 nM). Thermodynamic and kinetic Ca²⁺ binding studies showed that BP2 addition increased the Ca²⁺-binding affinity of CaM and slowed its Ca²⁺ dissociation rates (k_off) in both the C- and N-lobe EF-hand domains, respectively. These data are consistent with BP2 binding to the C-lobe of CaM at low free Ca²⁺ concentrations (<100 nM) like those found at resting intracellular levels. As free Ca²⁺ levels approach 1000 nM, which is typical inside a cell upon an intracellular Ca²⁺-signaling event, BP2 is shown here to interact with both the N- and C-lobes of Ca²⁺-loaded CaM (^CaCaM-BP2). Because this structural rearrangement observed for the ^CaCaM-BP2 complex occurs as intracellular free Ca²⁺ concentrations approach those typical of a Ca²⁺-signaling event (^CaK_D = 1000 ± 160 nM), this conformational change could be relevant to vitamin A transport by full-length ^CaCaM-STRA6.     

Ca2+ and Mg2+ modulate conformational dynamics and stability of downstream regulatory element antagonist modulator

Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca²⁺ concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca²⁺/Mg²⁺ association to DREAM. Our data indicate a minor impact of Ca²⁺ association on the overall structure of the N- and C-terminal domains, although Ca²⁺ binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca²⁺ bound forms of the protein. Time-resolved fluorescence data indicate that Ca²⁺binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca²⁺ association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca²⁺ and Mg²⁺ suggesting that, under physiological conditions, Ca²⁺ free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.     

Calcium binding to cardiac troponin C

The binding of Ca²⁺ to cardiac troponin C was studied by determining changes in the fluorescence and circular dichroism of the protein and by following changes in the free Ca²⁺ concentration by means of a Ca²⁺-specific electrode. Cardiac troponin C contains three Ca²⁺-binding sites which fall into two classes —two sites with a higher affinity and one with a lower affinity. The higher-affinity sites also bind Mg²⁺ which competes with the Ca²⁺.     

Equilibrium Dialysis Measurements of the Ca2+-Binding Properties of Recombinant Radish Vacuolar Ca2+-Binding Protein Expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca²⁺-binding protein (RVCaB) of 43 kDa. We investigated the Ca²⁺-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca²⁺-binding properties of the recombinant protein were examined by equilibrium dialysis with ⁴⁵Ca²⁺ and small dialysis buttons. The protein was estimated to bind 19Ca²⁺ ions per molecule with a K _d for Ca²⁺ of 3.4 mM. Ca²⁺ was bound to the protein even in the presence of high concentrations of Mg²⁺ or K⁺. The results suggested that the protein bound Ca²⁺ with high ion selectivity, high capacity, and low affinity.     

Membrane targeting of the EF-hand containing calcium-sensing proteins CaBP7 and CaBP8

The CaBP family of EF-hand containing small Ca²⁺-binding proteins have recently emerged as important regulators of multiple targets essential to normal neuronal function in the mammalian central nervous system. Of particular interest are CaBP7 and CaBP8, abundantly expressed brain proteins that exhibit the greatest sequence divergence from other family members. In this study, we have analysed their sub-cellular localisations in a model neuronal (Neuro2A) cell line and show that both proteins exhibit a membrane distribution distinct from the other CaBPs and consistent with localisation to the trans-Golgi network (TGN). Furthermore, we show that their localisation to the TGN critically depends upon an unusual predicted C-terminal transmembrane domain that if deleted or disrupted has dramatic consequences for protein targeting. CaBP7 and 8, therefore, possess a targeting mechanism that is unique amongst the CaBPs that may contribute to differential functional Ca²⁺-sensing by these family members.     

Solution NMR Structure of the Ca2+-bound N-terminal Domain of CaBP7: A REGULATOR OF GOLGI TRAFFICKING*

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-β (PI4KIIIβ) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIβ. CaBP7 NTD encompasses the two high affinity Ca²⁺ binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca²⁺ but not Mg²⁺ and undergoes significant conformational changes in both secondary and tertiary structure upon Ca²⁺ binding. The Ca²⁺-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca²⁺-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIβ.     

Alternative Splicing,Expression and Cellular Localization of Calneuron-1 in the Rat and Human Brain

Calneuron-1 and -2 are members of the neuronal calcium-binding protein family (nCaBP). They are transmembrane Calmodulin-like EF-hand Ca²⁺-sensors, and a function in the control of Golgi-to-plasma membrane vesicle trafficking has been assigned to both proteins. In this paper, we describe the distribution of Calneuron-1 in rat and human brains. We show that Calneuron-1 is ubiquitously expressed in all brain regions examined. The protein is most abundant in Purkinje cells of the cerebellum and principal neurons of the cortex and limbic brain whereas no expression in glial cells is apparent. In addition, we identify two novel splice isoforms of Calneuron-1 with extended N-termini. These isoforms are particular abundant in the cerebellum. Taken together, these data set grounds for a better understanding of the cellular function of Calneurons.     

A Highlights from MBoC Selection: Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

C2 domains are widespread motifs that often serve as Ca²⁺-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca²⁺ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca²⁺, and shifted the Ca²⁺ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.