penta-EF-hand Proteins Research Articles

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein–protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.

A second binding site revealed by C-terminal truncation of calpain small subunit, a penta-EF-hand protein.

The subunits in calpain and in the related penta-EF-hand (PEF) proteins are bound through contacts between the unpaired EF-hand 5 from each subunit. To study subunit binding further, a tetra-EF-hand 18 kDa N- and C-terminally truncated form of the calpain small subunit was prepared (18k). This protein does not combine with the calpain large subunit to form active calpain, but forms homodimers in solution, as shown by ultracentrifugation. The X-ray structure of the 18k protein in the presence of cadmium was solved to a resolution of 2.0 A. The structure of the monomer is almost identical to the known structure of the calpain small subunit, but the 18k protein forms an oligomer in the crystal by the use of two binding sites. One of these sites is an artefact arising from the C-terminal truncation, but the other is a naturally occurring site that is fully exposed to water in intact purified calpain. The characteristics of this site suggest that it may be important in binding other protein modulators involved in the regulation of calpain and of PEF proteins.

The penta-EF-hand domain of ALG-2 interacts with amino-terminal domains of both annexin VII and annexin XI in a Ca 2+-dependent manner

The apoptosis-linked protein ALG-2 is a Ca 2+-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca 2+-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST–Anx7N and GST–Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2–ΔN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants ( K d) were estimated to be approximately 40–60 nM for the high-affinity site and 500–700 nM for the low-affinity site.

The crystal structure of the sorcin calcium binding domain provides a model of Ca 2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca 2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca 2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca 2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH. The sorcin calcium binding domain (SCBD, residues 33–198) expressed in Escherichia coli was crystallized in the Ca 2+-free form. The structure was solved by molecular replacement and was refined to 2.2 Å with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers. The structure of the SCBD leads to a model that explains the solution properties and describes the Ca 2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca 2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca 2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca 2+-dependent binding to target proteins at or near membranes.

Both ALG-2 and Peflin, Penta-EF-hand (PEF) Proteins, Are Stabilized by Dimerization through Their Fifth EF-Hand Regions

ALG-2 (apoptosis-linked gene-2 protein) and peflin are Ca2+-binding proteins and belong to the penta-EF-hand (PEF) protein family, which includes calpain, sorcin, and grancalcin. ALG-2 forms either a homodimer or a heterodimer with peflin like other PEF proteins. In this study, we found that the fifth-EF-hand (EF-5) regions of both ALG-2 and peflin are essential for dimerization and their stabilities. Exogenously expressed EF-5-deletion (ΔEF-5) mutants of ALG-2 and peflin were unstable and were not detected in HEK293 cells by Western blotting. In a pulse–chase experiment, the ΔEF-5 mutants were rapidly degraded, but they were stabilized by treatment with a proteasome inhibitor, MG132. In MG132-treated cells, ΔEF-5 mutants were recovered in the insoluble fractions. Transient coexpression of ALG-2 increased the peflin level. These results indicate that the absence of a fifth EF-hand results in rapid degradation by the proteasome. On the other hand, stable expression of exogenous peflin decreased the amount of endogenous peflin. The amount of peflin that can dimerize with ALG-2 seems to be restricted in mammalian cells.

Crystal structure of calcium-free human sorcin: a member of the penta-EF-hand protein family.

Sorcin is a 22 kD calcium-binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta-EF-hand (PEF) protein family, which contains five EF-hand motifs that associate with membranes in a calcium-dependent manner. Prototypic members of this family are the calcium-binding domains of calpain, such as calpain dVI. Full-length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 A resolution. Apart from an extended N-terminal portion, the sorcin molecule has a globular shape. The C-terminal domain is predominantly alpha-helical, containing eight alpha-helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF-hand motifs are conserved, especially that of EF1, the novel EF-hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF-hand pair EF1-EF2 is likely to correspond to the two physiologically relevant calcium-binding sites and that the calcium-induced conformational change may be modest and localized within this pair of EF-hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF-hands.

Identification and characterization of two penta-EF-hand Ca(2+)-binding proteins in Dictyostelium discoideum.

Penta-EF-hand (PEF) proteins such as ALG-2 (apoptosis-linked gene 2 product) and the calpain small subunit are a newly classified family of Ca(2+)-binding proteins that possess five EF-hand-like motifs. We identified two mutually homologous PEF proteins, designated DdPEF-1 and DdPEF-2 (64% amino acid residue identities), in the cellular slime mold Dictyostelium discoideum. Both PEF proteins showed a higher similarity to mammalian ALG-2 and peflin (Group I PEF proteins) than to calpain and sorcin subfamily (Group II PEF proteins) in the first EF-hand (EF-1) regions. Northern blot analyses revealed that DdPEF-1 and DdPEF-2 were constitutively expressed throughout development of Dictyostelium, but their levels of expression were developmentally regulated. In situ hybridization analyses demonstrated that DdPEF-1 was expressed in both the anterior prestalk and the posterior prespore regions of the tipped aggregate, slugs and early culminants. On the other hand, DdPEF-2 was dominantly expressed in the anterior tip region of these multicellular structures. Both PEF proteins were detected as 22-23-kDa proteins in soluble fractions in the presence of EGTA but in particulate fractions in the presence of Ca(2+) by Western blotting using specific monoclonal antibodies. Together with the finding of PEF-like sequences in DNA databases of plants, fungi and protists, our results strongly suggest that Group I PEF proteins are ubiquitously present in all eukaryotes and play important roles in basic cellular functions.

Peflin and ALG-2, Members of the Penta-EF-Hand Protein Family, Form a Heterodimer That Dissociates in a Ca2+-dependent Manner

Peflin, a newly identified 30-kDa Ca(2+)-binding protein, belongs to the penta-EF-hand (PEF) protein family, which includes the calpain small subunit, sorcin, grancalcin, and ALG-2 (apoptosis-linked gene 2). We prepared a monoclonal antibody against human peflin. The antibody immunoprecipitated a 22-kDa protein as well as the 30-kDa protein from the lysate of Jurkat cells. Western blotting of the immunoprecipitates revealed that the 22-kDa protein corresponds to ALG-2. This was confirmed by Western blotting of the immunoprecipitates of epitope-tagged peflin or ALG-2 whose cDNA expression constructs were transfected to human embryonic kidney (HEK) 293 cells. Gel filtration of the cytosolic fraction of Jurkat cells revealed co-elution of peflin and ALG-2 in fractions eluting earlier than recombinant ALG-2, further supporting the notion of heterodimerization of the two PEF proteins. Surprisingly, peflin dissociated from ALG-2 in the presence of Ca(2+). Peflin and ALG-2 co-localized in the cytoplasm, but ALG-2 was also detected in the nuclei as revealed by immunofluorescent staining and subcellular fractionation. Peflin was recovered in the cytosolic fraction in the absence of Ca(2+) but in the membrane/cytoskeletal fraction in the presence of Ca(2+). These results suggest that peflin has features common to those of other PEF proteins (dimerization and translocation to membranes) and may modulate the function of ALG-2 in Ca(2+) signaling.

Structure of Apoptosis-Linked Protein ALG-2: Insights into Ca 2+-Induced Changes in Penta-EF-Hand Proteins

Background: The Ca 2+ binding apoptosis-linked gene-2 (ALG-2) protein acts as a proapoptotic factor in a variety of cell lines and is required either downstream or independently of caspases for apoptosis to occur. ALG-2 belongs to the penta-EF-hand (PEF) protein family and has two high-affinity and one low-affinity Ca 2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca 2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). Results: We present the 2.3 Å resolution crystal structure of Ca 2+-loaded des1-20ALG-2 (aa 21–191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight α helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca 2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca 2+ and is thought to primarily facilitate dimerization. Most importantly, the conformation of des1-20ALG-2 is significantly different from that of calpain and grancalcin. This difference can be described as a rigid body rotation of EF1-2 relative to EF4-5 and the dimer interface, with a hinge within the EF3 loop. An electron density, which is interpreted as a hydrophobic Gly/Pro-rich decapeptide that is possibly derived from the cleaved N terminus, was found in a hydrophobic cleft between these two halves of the molecule. Conclusions: A different relative orientation of the N- and C-terminal halves of des1-20ALG-2 in the presence of Ca 2+ and the peptide as compared to other Ca 2+-loaded PEF proteins changes substantially the shape of the molecule, exposing a hydrophobic patch on the surface for peptide binding and a large cleft near the dimer interface. We postulate that the binding of a Gly/Pro-rich peptide in the presence of Ca 2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.