Origin of the NEFA and Nuc Signal Sequences

The human protein NEFA binds calcium, contains a leucine zipper repeat that does not form a homodimer, and is proposed (along with the homologous Nuc protein) to have a common evolutionary history with an EF-hand ancestor. We have isolated and characterized the N-terminal domain of NEFA that contains a signal sequence inferred from both endoproteinase Asp-N (Asp-N) and tryptic digests. Analysis of this N-terminal sequence shows significant similarity to the conserved multiple domains of the mitochondrial carrier family (MCF) proteins. The leader sequence of Nuc is, however, most similar to the signal sequences of membrane and/or secreted proteins (e.g., mouse insulin-like growth factor receptor). We suggest that the divergent NEFA and Nuc N-terminal sequences may have independent origins and that the common high hydrophobicity governs their targeting to the ER. These results provide insights into signal sequence evolution and the multiple origins of protein targeting. Received: 20 February 1997 / Accepted: 28 July 1997     

Heterologous overexpression of human NEFA and studies on the two EF-hand calcium-binding sites.

Human NEFA is an EF-hand, leucine zipper protein containing a signal sequence. To confirm the calcium binding capacity of NEFA, recombinant NEFA analogous to the mature protein and mutants with deletions in the EF-hand domain were expressed in Pichia pastoris and secreted into the culture medium at high yield. The calcium binding activity of each purified protein was measured by a modified equilibrium dialysis using the fluorescent Ca2+ indicator FURA-2 and atomic absorption spectroscopy. A stoichiometry of 2 mol Ca2+/mol NEFA was determined. The Ca2+ binding constants were resolved by intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca2+ binding sites with Kd values of 0.08 microM and 0.2 microM. Circular dichroism (CD) spectroscopy showed an increase from 30 to 43% in the amount of alpha-helix in NEFA after addition of calcium ions. Limited proteolytic digestion indicated a Ca2+ dependent conformational change accompanied by an altered accessibility to the enzyme.     

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.     

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.

The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.     

Conformation and structural transitions in the EF-hands of calmodulin

Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.     

Structures and metal-ion-binding properties of the Ca2+-binding helix-loop-helix EF-hand motifs

The 'EF-hand' Ca2+-binding motif plays an essential role in eukaryotic cellular signalling, and the proteins containing this motif constitute a large and functionally diverse family. The EF-hand is defined by its helix-loop-helix secondary structure as well as the ligands presented by the loop to bind the Ca2+ ion. The identity of these ligands is semi-conserved in the most common (the 'canonical') EF-hand; however, several non-canonical EF-hands exist that bind Ca2+ by a different co-ordination mechanism. EF-hands tend to occur in pairs, which form a discrete domain so that most family members have two, four or six EF-hands. This pairing also enables communication, and many EF-hands display positive co-operativity, thereby minimizing the Ca2+ signal required to reach protein saturation. The conformational effects of Ca2+ binding are varied, function-dependent and, in some cases, minimal, but can lead to the creation of a protein target interaction site or structure formation from a molten-globule apo state. EF-hand proteins exhibit various sensitivities to Ca2+, reflecting the intrinsic binding ability of the EF-hand as well as the degree of co-operativity in Ca2+ binding to paired EF-hands. Two additional factors can influence the ability of an EF-hand to bind Ca2+: selectivity over Mg2+ (a cation with very similar chemical properties to Ca2+ and with a cytoplasmic concentration several orders of magnitude higher) and interaction with a protein target. A structural approach is used in this review to examine the diversity of family members, and a biophysical perspective provides insight into the ability of the EF-hand motif to bind Ca2+ with a wide range of affinities.     

LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.

Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.     

Dimerization of cGMP-dependent protein kinase 1alpha and the myosin-binding subunit of myosin phosphatase: role of leucine zipper domains

Nitric oxide (NO) and nitrovasodilators induce vascular smooth muscle cell relaxation in part by cGMP-dependent protein kinase (cGK)-mediated activation of myosin phosphatase, which dephosphorylates myosin light chains. We recently found that cGMP-dependent protein kinase 1alpha binds directly to the myosin-binding subunit (MBS) of myosin phosphatase via the leucine/isoleucine zipper of cGK. We have now studied the role of the leucine zipper domain of MBS in dimerization with cGK and the leucine/isoleucine zipper and leucine zipper domains of both proteins in homodimerization. Mutagenesis of the MBS leucine zipper domain disrupts cGKIalpha-MBS dimerization. Mutagenesis of the MBS leucine zipper eliminates MBS homodimerization, while similar disruption of the cGKIalpha leucine/isoleucine zipper does not prevent formation of cGK dimers. The MBS leucine zipper domain is phosphorylated by cGK, but this does not have any apparent effect on heterodimer formation between the two proteins. MBS LZ mutants that are unable to bind cGK were poor substrates for cGK. These data support the theory that the MBS leucine zipper domain is necessary and sufficient to mediate both MBS homodimerization and binding of the protein to cGK. In contrast, the leucine/isoleucine zipper of cGK is required for binding to MBS, but not for cGK homodimerization. These data support that the MBS and cGK leucine zipper domains mediate the interaction between these two proteins. The contribution of these domains to both homodimerization and their specific interaction with each other suggest that additional regulatory mechanisms involving these domains may exist.     

Binding of guanylyl cyclase activating protein 1 (GCAP1) to retinal guanylyl cyclase (RetGC1). The role of individual EF-hands

Guanylyl cyclase activating protein 1 (GCAP1), after substitution of Ca(2+) by Mg(2+) in its EF-hands, stimulates photoreceptor guanylyl cyclase, RetGC1, in response to light. We inactivated metal binding in individual EF-hands of GCAP1 tagged with green fluorescent protein to assess their role in GCAP1 binding to RetGC1 in co-transfected HEK293 cells. When expressed alone, GCAP1 was uniformly distributed throughout the cytoplasm and the nuclei of the cells, but when co-expressed with either fluorescently tagged or non-tagged RetGC1, it co-localized with the cyclase in the membranes. The co-localization did not occur when the C-terminal portion of RetGC1, containing its regulatory and catalytic domains, was removed. Mutations that preserved Mg(2+) binding in all three metal-binding EF-hands did not affect GCAP1 association with the cyclase in live cells. Locking EF-hand 4 in its apo-conformation, incapable of binding either Ca(2+) or Mg(2+), had no effect on GCAP1 association with the cyclase. In contrast to EF-hand 4, inactivation of EF-hand 3 reduced the efficiency of the co-localization, and inactivation of EF-hand 2 drastically suppressed GCAP1 binding to the cyclase. These results directly demonstrate that metal binding in EF-hand 2 is crucial for GCAP1 attachment to RetGC1, and that in EF-hand 3 it is less critical, although it enhances the efficiency of the GCAP1 docking on the target enzyme. Metal binding in EF-hand 4 has no role in the primary attachment of GCAP1 to the cyclase, and it only triggers the activator-to-inhibitor functional switch in GCAP1.     

Calbindin D28k EF-hand ligand binding and oligomerization: four high-affinity sites--three modes of action

Calbindin D28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Ca2+ with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between the EF-hand subdomains. In this study, we have undertaken a detailed characterization of the Ca2+-binding and oligomerization properties of each individual EF-hand peptide using CD spectroscopy and analytical ultracentrifugation. Under the conditions tested, EF2 is monomeric and does not bind Ca2+, whereas EF6, which binds Ca2+ weakly, aggregates severely. We have therefore focused this study on the high-affinity binding sites, EF-hands 1, 3, 4, and 5. Our sedimentation equilibrium data show that, in the presence of Ca2+, EF-hands 1, 3, 4, and 5 all form dimers in solution in which the distribution between the monomer, dimer, and higher order oligomers differs. The processes of Ca2+ binding and oligomerization are linked to different degrees, and three main mechanisms emerge. For EF-hands 1 and 5, the dimer binds Ca2+ more strongly than the monomer and Ca2+ binding drives dimerization. For EF-hand 4, dimer formation requires only one of the monomers to be Ca2+-bound. In this case, the Ca2+ affinity is independent of dimerization. For EF-hand 3, dimerization occurs both in the absence and presence of Ca2+, while oligomerization increases in the presence of Ca2+.     

Structural characterization of calcineurin B homologous protein 1

Calcineurin B homologous protein 1 (CHP1), also known as p22, is a calcium-binding EF-hand protein that plays a role in membrane trafficking. It binds to multiple effector proteins, including Na(+)/H(+) exchangers, a serine/threonine kinase, and calcineurin, potentially modulating their function. The crystal structure of calcium-bound CHP1 from rat has been determined at 2.2 Angstroms of resolution. The molecule has a compact alpha-helical structure containing four EF-hands. The overall folding topology of the protein is similar to that of the regulatory B subunit of calcineurin and to that of calcium- and integrin-binding protein. The calcium ion is coordinated in typical fashion in the third and fourth EF-hands, but the first and second EF-hands contain no calcium ion. The first EF-hand is maintained by internal interactions, and the second EF-hand is stabilized by hydrophobic interactions. CHP1 contains a hydrophobic pocket on the opposite side of the protein to the EF-hands that has been implicated in ligand binding.     

A molecular clasp in the human immunodeficiency virus (HIV) type 1 TM protein determines the anti-HIV activity of gp41 derivatives: implication for viral fusion.

We have previously reported that synthetic peptides representing the leucine zipper domain (DP107) and a second putative helical domain (DP178) of human immunodeficiency virus type 1 (HIV-1) gp41 exhibit potent anti-HIV activity. In this study we have used soluble recombinant forms of gp41 to provide evidence that the DP178 peptide and the DP178 region of gp41 associate with a distal site on the gp41 transmembrane protein whose interactive structure is influenced by the leucine zipper (DP107) motif. We also observed that a single coiled-coil-disrupting mutation in the leucine zipper domain transformed the recombinant gp41 protein from an inactive to an active inhibitor of HIV-1 fusion and infectivity, which may be related to that finding. We speculate that this transformation results from liberation of the potent DP178-related sequence from a molecular clasp with a leucine zipper, DP107, determinant. The results are discussed in the context of two distinct conformations for the gp41 molecule and possible involvement of these two domains in structural transitions associated with HIV-1-mediated fusion. The results are also interpreted to suggest that the anti-HIV activity of the various gp41 derivatives (peptides and recombinant proteins) may be due to their ability to form complexes with viral gp41 and interfere with its fusogenic processes.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein

Trypanosoma brucei BILBO1 (TbBILBO1) is an essential component of the flagellar pocket collar of trypanosomes. We recently reported the high resolution structure of the N-terminal domain of TbBILBO1. Here, we provide further structural dissections of its other three constituent domains: EF-hand, coiled coil, and leucine zipper. We found that the EF-hand changes its conformation upon calcium binding, the central coiled coil forms an antiparallel dimer, and the C-terminal leucine zipper appears to contain targeting information. Furthermore, interdimer interactions between adjacent leucine zippers allow TbBILBO1 to form extended filaments in vitro. These filaments were additionally found to condense into fibers through lateral interactions. Based on these experimental data, we propose a mechanism for TbBILBO1 assembly at the flagellar pocket collar.     

Coupling of ligand binding and dimerization of helix-loop-helix peptides: spectroscopic and sedimentation analyses of calbindin D9k EF-hands

Isolated Ca2+-binding EF-hand peptides have a tendency to dimerize. This study is an attempt to account for the coupled equilibria of Ca2+-binding and peptide association for two EF-hands with strikingly different loop sequence and net charge. We have studied each of the two separate EF-hand fragments from calbindin D9k. A series of Ca2+-titrations at different peptide concentrations were monitored by CD and fluorescence spectroscopy. All data were fitted simultaneously to both a complete model of all possible equilibrium intermediates and a reduced model not including dimerization in the absence of Ca2+. Analytical ultracentrifugation shows that the peptides may occur as monomers or dimers depending on the solution conditions. Our results show strikingly different behavior for the two EF-hands. The fragment containing the N-terminal EF-hand shows a strong tendency to dimerize in the Ca2+-bound state. The average Ca2+-affinity is 3.5 orders of magnitude lower than for the intact protein. We observe a large apparent cooperativity of Ca2+ binding for the overall process from Ca2+-free monomer to fully loaded dimer, showing that a Ca2+-free EF-hand folds upon dimerization to a Ca2+-bound EF-hand, thereby presenting a preformed binding site to the second Ca2+-ion. The C-terminal EF-hand shows a much smaller tendency to dimerize, which may be related to its larger net negative charge. In spite of the differences in dimerization behavior, the Ca2+ affinities of both EF-hand fragments are similar and in the range lgK = 4.6-5.3.     

The B cell receptor-induced calcium flux involves a calcium mediated positive feedback loop

The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells.     

Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+/Ca2+ exchange in EF-hand domains

Guanylyl cyclase activating protein 1 (GCAP-1), a Ca(2+)/Mg(2+) sensor protein that accelerates retinal guanylyl cyclase (RetGC) in the light and decelerates it in the dark, is inactive in cation-free form. Binding of Mg(2+) in EF-hands 2 and 3 was essential for RetGC activation in the conditions mimicking light adaptation. Mg(2+) binding in EF-hand 2 affected the conformation of a neighboring non-metal binding domain, EF-hand-1, and increased GCAP-1 affinity for RetGC nearly 40-fold compared with the metal-free EF-hand 2. Mg(2+) binding in EF-hand 3 increased GCAP-1 affinity for RetGC 5-fold and its maximal RetGC stimulation 2-fold. Mg(2+) binding in EF-hand 4 affected neither GCAP-1 affinity for RetGC, nor RetGC activation. Inactivation of Ca(2+) binding in EF-hand 4 was sufficient to render GCAP-1 a constitutive activator of RetGC, whereas the EF-hand 3 role in Ca(2+)-dependent deceleration of RetGC was likely to be through the neighboring EF-hand 4. Inactivation of Ca(2+) binding in EF-hand 2 affected cooperativity of RetGC inhibition by Ca(2+), but did not prevent the inhibition. We conclude that 1) Mg(2+) binding in EF-hands 2 and 3, but not EF-hand 4, is essential for the ability of GCAP-1 to activate RetGC in the light; 2) Mg(2+) or Ca(2+) binding in EF-hand 3 and especially in EF-hand 2 is required for high-affinity interaction with the cyclase and affects the conformation of the neighboring EF-hand 1, a domain required for targeting RetGC; and 3) RetGC inhibition is likely to be primarily caused by Ca(2+) binding in EF-hand 4.