Analysis of EF-hand-containing proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Background  

In plants, calcium (Ca²⁺) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca²⁺]_cyt), which in turn is thought to regulate cellular and developmental processes via Ca²⁺-binding proteins. Three out of the four classes of Ca²⁺-binding proteins in plants contain Ca²⁺-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca²⁺ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.     

NMR structure and calcium-binding properties of the tellurite resistance protein TerD from Klebsiella pneumoniae

The tellurium oxyanion TeO₃²⁻ has been used in the treatment of infectious diseases caused by mycobacteria. However, many pathogenic bacteria show tellurite resistance. Several tellurite resistance genes have been identified, and these genes mediate responses to diverse extracellular stimuli, but the mechanisms underlying their functions are unknown. To shed light on the function of KP-TerD, a 20.5 -kDa tellurite resistance protein from a plasmid of Klebsiella pneumoniae, we have determined its three-dimensional structure in solution using NMR spectroscopy. KP-TerD contains a β-sandwich formed by two five-stranded β-sheets and six short helices. The structure exhibits two negative clusters in loop regions on the top of the sandwich, suggesting that KP-TerD may bind metal ions. Indeed, thermal denaturation experiments monitored by circular dichroism and NMR studies reveal that KP-TerD binds Ca²⁺. Inductively coupled plasma-optical emission spectroscopy shows that the binding ratio of KP-TerD to Ca²⁺ is 1:2. EDTA (ethylenediaminetetraacetic acid) titrations of Ca²⁺-saturated KP-TerD monitored by one-dimensional NMR yield estimated dissociation constants of 18  and 200 nM for the two Ca²⁺-binding sites of KP-TerD. NMR structures incorporating two Ca²⁺ ions define a novel bipartite Ca²⁺-binding motif that is predicted to be highly conserved in TerD proteins. Moreover, these Ca²⁺-binding sites are also predicted to be present in two additional tellurite resistance proteins, TerE and TerZ. These results suggest that some form of Ca²⁺ signaling plays a crucial role in tellurite resistance and in other responses of bacteria to multiple external stimuli that depend on the Ter genes.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

Molecular Tuning of an EF-Hand-like Calcium Binding Loop : Contributions of the Coordinating Side Chain at Loop Position 3

Calcium binding and signaling orchestrate a wide variety of essential cellular functions, many of which employ the EF-hand Ca²⁺ binding motif. The ion binding parameters of this motif are controlled, in part, by the structure of its Ca²⁺ binding loop, termed the EF-loop. The EF-loops of different proteins are carefully specialized, or fine-tuned, to yield optimized Ca²⁺ binding parameters for their unique cellular roles. The present study uses a structurally homologous Ca²⁺ binding loop, that of the Escherichia coli galactose binding protein, as a model for the EF-loop in studies examining the contribution of the third loop position to intramolecular tuning. 10 different side chains are compared at the third position of the model EF-loop with respect to their effects on protein stability, sugar binding, and metal binding equilibria and kinetics. Substitution of an acidic Asp side chain for the native Asn is found to generate a 6,000-fold increase in the ion selectivity for trivalent over divalent cations, providing strong support for the electrostatic repulsion model of divalent cation charge selectivity. Replacement of Asn by neutral side chains differing in size and shape each alter the ionic size selectivity in a similar manner, supporting a model in which large-ion size selectivity is controlled by complex interactions between multiple side chains rather than by the dimensions of a single coordinating side chain. Finally, the pattern of perturbations generated by side chain substitutions helps to explain the prevalence of Asn and Asp at the third position of natural EF-loops and provides further evidence supporting the unique kinetic tuning role of the gateway side chain at the ninth EF-loop position.     

Amino acid sequence of squid troponin C

The complete amino acid sequence of squid Todarodes pacificus troponin C (TnC), which was shown to bind only 1 mol Ca²⁺/mol, was determined by both the Edman and cDNA methods. The squid TnC is composed of 147 amino acids including an unblocked Pro at the N-terminus and the calculated molecular weight is 17 003.9. Among the four potential Ca²⁺-binding sites, namely sites I–IV from the N-terminus, only site IV completely satisfied the consensus amino acid sequence for the active Ca²⁺-binding loop. This indicates that squid TnC possesses a single Ca²⁺-binding site at the site IV as scallop TnCs [Nishita et al., J. Biol. Chem. 269 (1994) 3464–3468; Ojima et al., Arch. Biochem. Biophys. 311 (1994) 272–276). The sequence homology of squid TnC to TnCs of scallop, arthropods, and rabbit was 61%, 31–38%, and 31%, respectively. In the sequence of the central D/E-helix region of squid and scallop TnCs, a deletion of three amino acids was required to maximize the homology with the other TnCs.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.     

Pf1 virus particle dynamics

The overall dynamics of the Pf1 filamentous bacteriophage particle in solution are characterized by nmr experiments. The chemical-shift anisotropy powder-pattern lineshapes from both DNA and protein backbone sites of the virus are motionally averaged in the same way, indicating that the entire particle undergoes rapid (< 10⁴ Hz) reorientation about the long axis of the filament when the virus is in solution at high pH. In contrast, the virus particles in samples at low pH are immobile on this time scale.     

Penta-EF-hand protein ALG-2 functions as a Ca-dependent adaptor that bridges Alix and TSG101

Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca²⁺-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca²⁺-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca²⁺-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.     

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.     

Calmodulin Mediates the Ca-Dependent Regulation of Cx44 Gap Junctions

We have shown previously that the Ca²⁺-dependent inhibition of lens epithelial cell-to-cell communication is mediated in part by the direct association of calmodulin (CaM) with connexin43 (Cx43), the major connexin in these cells. We now show that elevation of [Ca²⁺]_i in HeLa cells transfected with the lens fiber cell gap junction protein sheep Cx44 also results in the inhibition of cell-to-cell dye transfer. A peptide comprising the putative CaM binding domain (aa 129-150) of the intracellular loop region of this connexin exhibited a high affinity, stoichiometric interaction with Ca²⁺-CaM. NMR studies indicate that the binding of Cx44 peptide to CaM reflects a classical embracing mode of interaction. The interaction is an exothermic event that is both enthalpically and entropically driven in which electrostatic interactions play an important role. The binding of the Cx44 peptide to CaM increases the CaM intradomain cooperativity and enhances the Ca²⁺-binding affinities of the C-domain of CaM more than twofold by slowing the rate of Ca²⁺ release from the complex. Our data suggest a common mechanism by which the Ca²⁺-dependent inhibition of the α-class of gap junction proteins is mediated by the direct association of an intracellular loop region of these proteins with Ca²⁺-CaM.     

Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca²⁺ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca²⁺-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca²⁺-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca²⁺-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca²⁺-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca²⁺-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca²⁺ signal to TnI in Tn ternary complex.     

Inhibition of the Ca2+-and phospholipid-dependent protein kinase by a novel Mr 17,000 Ca2+-binding protein

A novel M_r 17,000 Ca²⁺-binding protein isolated from bovine brain was found to be a potent inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase (protein kinase C), also isolated from bovine brain. Halfmaximal inhibition by this calciprotein of the initial rate of phosphorylation of histone III-S by protein kinase C occurred at a calciprotein concentration of 2.2 μM under standard conditions. Comparison of the effects of a number of Ca²⁺-binding proteins on protein kinase C activity indicated that the M_r 17,000 Ca²⁺-binding protein was the most potent inhibitor, followed by the intestinal Ca²⁺-binding protein and calcineurin. Calmodulin, troponin C, S-100 protein and a M_r 21,000 Ca²⁺-binding protein of bovine brain were relatively weak inhibitors of protein kinase C. The inhibitory effect of the M_r 17,000 Ca²⁺-binding protein was apparently not due to its interaction with phospholipid or the basic protein substrate and therefore appears to be due to a direct effect on the protein kinase C. These observations suggest that the novel M_r 17,000 Ca²⁺-binding protein, and possibly other Ca²⁺-binding proteins, may play a physiological role in regulating the activity of protein kinase C.     

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca²⁺ but to a lesser extent (2–3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca²⁺ binding to the C-terminal domain of CaM with an associated increase in the Ca²⁺ dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca²⁺ binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca²⁺-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca²⁺ binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca²⁺-bound CaM and that although Ca²⁺ binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca²⁺ binding to the C-terminal lobe of CaM.     

Calcium-regulated GTPase activity in the calcium-binding protein calexcitin

Calexcitin (CE) is a calcium-binding protein, closely related to sarcoplasmic calcium-binding proteins, that is involved in invertebrate learning and memory. Early reports indicated that both Hermissenda and squid CE also could bind GTP; however, the biochemical significance of GTP-binding and its relationship to calcium binding have remained unclear. Here, we report that the GTPase activity of CE is strongly regulated by calcium. CE possessed a P-loop-like structure near the C-terminal similar to the phosphate-binding regions in other GTP-binding proteins. Site-directed mutagenesis of this region showed that Gly¹⁸², Phe¹⁸⁶ and Gly¹⁸⁷ are required for maximum affinity, suggesting that the GTP-binding motif is G-N-x-x-[FM]-G. CE cloned from Drosophila CNS possessed a similar C-terminal sequence and also bound and hydrolyzed GTP. GTPase activity in Drosophila CE was also strongly regulated by Ca²⁺, exhibiting over 23-fold higher activity in the presence of 0.3 μM calcium. Analysis of the conserved protein motifs defines a new family of Ca²⁺-binding proteins representing the first example of proteins endowed with both EF-hand calcium binding domains and a C-terminal, P-loop-like GTP-binding motif. These results establish that, in the absence of calcium, both squid and Drosophila CE bind GTP at near-physiological concentrations and hydrolyze GTP at rates comparable to unactivated ras. Calcium functions to increase GTP-binding and GTPase activity in CE, similar to the effect of GTPase activating proteins in other low-MW GTP-binding proteins. CE may, therefore, act as a molecular interface between Ca²⁺ cytosolic oscillations and the G protein-coupled signal transduction.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.