The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins

The structure of bovine intestinal calcium-binding protein (ICaBP) has been determined crystallographically at a resolution of 2.3 A and refined by a least squares technique to an R factor of 17.8%. The refined structure includes all 600 non-hydrogen protein atoms, two bound calcium ions, and solvent consisting of one sulfate ion and 36 water molecules. The molecule consists of two helix-loop-helix calcium-binding domains known as EF hands, connected by a linker containing a single turn of helix. Helix-helix interactions are primarily hydrophobic, but also include a few strategic hydrogen bonds. Most of the hydrogen bonds, however, are found in the calcium-binding loops, where they occur both within a single loop and between the two. Examination of the hydrogen bonding patterns in the calcium-binding loops of ICaBP and the related protein, parvalbumin, reveals several conserved hydrogen bonds which are evidently important for loop stabilization. The primary and tertiary structural features which promote the formation of an EF hand were originally identified from the structure of parvalbumin. They are modified in light of the ICaBP structure and considered as they apply to other calcium-binding proteins. The C-terminal domain of ICaBP is a normal EF hand, with ion binding properties similar to those of the calmodulin hands, but the N-terminal domain is a variant hand whose calcium ligands are mostly peptide carbonyls. Relative to a normal EF hand, this domain exhibits a similar KD for calcium binding but a greatly reduced affinity for calcium analogs such as cadmium and the lanthanide series. Lanthanides in particular may be inappropriate models for calcium in this system.     

Structure of the neuronal protein calexcitin suggests a mode of interaction in signalling pathways of learning and memory

The three-dimensional structure of the neuronal calcium-sensor protein calexcitin from Loligo pealei has been determined by X-ray analysis at a resolution of 1.8A. Calexcitin is up-regulated following Pavlovian conditioning and has been shown to regulate potassium channels and the ryanodine receptor. Thus, calexcitin is implicated in neuronal excitation and plasticity. The overall structure is predominantly helical and compact with a pronounced hydrophobic core between the N and C-terminal domains of the molecule. The structure consists of four EF-hand motifs although only the first three EF hands are involved in binding calcium ions; the C-terminal EF-hand lacks the amino acids required for calcium binding. The overall structure is quite similar to that of the sarcoplasmic calcium-binding protein from Amphioxus although the sequence identity is very low at 31%. The structure shows that the two amino acids of calexcitin phosphorylated by protein kinase C are close to the domain interface in three dimensions and thus phosphorylation is likely to regulate the opening of the domains that is probably required for binding to target proteins. There is evidence that calexcitin is a GTPase and the residues, which have been implicated by mutagenesis in its GTPase activity, are in a short but highly conserved region of 3(10) helix close to the C terminus. This helix resides in a large loop that is partly sandwiched between the N and C-terminal domains suggesting that GTP binding may also require or may cause domain opening. The structure possesses a pronounced electropositive crevice in the vicinity of the 3(10) helix, that might provide an initial docking site for the triphosphate group of GTP. These findings elucidate a number of the reported functions of calexcitin with implications for neuronal signalling.     

Effective association of Kv channel-interacting proteins with Kv4 channel is mediated with their unique core peptide

Kv channel-interacting proteins (KChIPs) and neuronal calcium sensor-1 (NCS-1) have been shown to interact with Kv4 channel alpha-subunits to regulate the expression and/or gating of these channels. Here we examine the specificity and sites of these proteins for interaction with Kv channel proteins. Immunoprecipitation and green fluorescent protein imaging show that KChIPs (but not NCS-1) effectively bind to Kv4.3 protein and localize at the plasma membrane when channel proteins are coexpressed. Analysis with chimeric proteins between KChIP2 and NCS-1 reveals that the three regions of KChIP2 (the linker between the first and second EF hands, the one between the third and fourth EF hands, and the C-terminal peptide after the fourth EF hand) are necessary and sufficient for its effective binding to Kv4.3 protein. The chimera with these three KChIP2 portions slowed inactivation and facilitated recovery from inactivation of Kv4.3 current. These results indicate that the sequence difference in these three regions between KChIPs and NCS-1 determines the specificity and affinity for interaction with Kv4 protein. Because the three identified regions surround the large hydrophobic crevice based on the NCS-1 crystal structure, this crevice may be the association site of KChIPs for the channel protein.     

Structural differences among subfamilies of EF‐hand proteins—A view from the pseudo two‐fold symmetry axis

We have analyzed the conformations of EF‐lobes, adjacent pairs of EF‐hand domains, in a coordinate system based on the approximate two‐fold (z) axis that relates the two EF‐hands. Two parameters ‐ dE(ø), the azimuthal angle between the y‐axis and the projection of the offset vector to helix E onto the yz‐plane, and δdF(ø), the difference angle between the two helices (F1 and F2) of odd and even domains—characterize the openness of a single EF‐hand domain and of an EF‐lobe, respectively. We describe and compare values of dE(ø) and of δdF(ø) for EF‐hand proteins of five subfamilies—CTER, CPV, S100, PARV, CALP—in calci‐ and apo‐ forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins. Proteins 2014; 82:2915–2924. © 2014 Wiley Periodicals, Inc.     

Contribution of the different modules in the utrophin carboxy-terminal region to the formation and regulation of the DAP complex

The carboxy-terminal region of utrophin, like the homologous proteins dystrophin, Drp2 and dystrobrevins, contains structural domains frequently involved in protein-protein interaction. These domains (WW, EF hands, ZZ and H1-H2) mediate recognition and binding to a multicomponent complex of proteins, also known as dystrophin-associated proteins (DAPs) for their association with dystrophin, the product of the gene, mutated in Duchenne muscular dystrophy. We have exploited phage display and in vitro binding assays to study the recognition specificity of the different domains of the utrophin carboxy-terminus. We found that none of the carboxy-terminal domains of utrophin, when isolated from its structural context, selects specific ligand peptides from a phage-displayed peptide library. By contrast, panning with an extended region containing the WW, EF hands, and ZZ domain defines the consensus binding motif, PPxY which is also found in beta-dystroglycan, a component of the DAP complex that interacts with utrophin in several tissues. WW-mediated binding to PPxY peptides and to beta-dystroglycan requires the presence of the EF hands and ZZ domain. When the ZZ domain is either deleted or engaged in binding to calmodulin, the utrophin beta-dystroglycan complex cannot be formed. These findings suggest a potential regulatory mechanism by means of which the attachment of utrophin to the DAP complex can be modulated by the Ca(2+)-dependent binding of calmodulin. The remaining two motifs found in the carboxy-terminus (H1-H2) mediate the formation of utrophin-dystrobrevin hybrids but do not select ligands in a repertoire of random nonapeptides.     

The C-terminal domain of pediocin-like antimicrobial peptides (class IIa bacteriocins) is involved in specific recognition of the C-terminal part of cognate immunity proteins and in determining the antimicrobial spectrum

The pediocin-like bacteriocins contain two domains: a cationic N-terminal beta-sheet domain that mediates binding of the bacteriocin to the target cell surface and a more hydrophobic C-terminal hairpin-like domain that penetrates into the hydrophobic part of the target cell membrane. The two domains are joined by a hinge, which enables movement of the domains relative to each other. In this study, 12 different hybrid bacteriocins were constructed by exchanging domains between 5 different bacteriocins. The hybrid bacteriocins were by and large highly potent (i.e. similar potencies as the parental bacteriocins) when constructed such that the recombination point was in the hinge region, indicating that the two domains function independently. The use of optimal recombination points was, however, crucial. Shifting the recombination point just one residue from the hinge could reduce the activity of the hybrid by 3-4 orders of magnitude. Most interestingly, the active hybrids displayed target cell specificities similar to those of the parental bacteriocin from which their membrane-penetrating C-terminal hairpin domain was derived. The results also indicate that the negatively charged aspartate reside in the hinge of most pediocin-like bacteriocins interacts with the C-terminal hairpin domain, perhaps by interacting with the positively charged residue that is present at one of the last three positions in the C-terminal end of most pediocin-like bacteriocins. Bacteria that produce pediocin-like bacteriocins also produce a cognate immunity protein that protects the producer from being killed by its own bacteriocin. Four different active hybrid immunity proteins constructed by exchanging regions between three different immunity proteins were tested for their ability to confer immunity to the hybrid bacteriocins. The results showed that the C-terminal half of the immunity proteins contains a region that directly or indirectly specifically recognizes the membrane-penetrating C-terminal hairpin domain of pediocin-like bacteriocins. The implications these results have on how pediocin-like bacteriocins and their immunity proteins interact with cellular specificity determinants (for instance a putative bacteriocin receptor) are discussed.     

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A 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 Ca2+-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 Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.     

Characterization of the Ca2+-switch in skeletal and cardiac muscles

To determine the significance of the global structure of the regulatory proteins in the mechanism of the Ca2+-switch in cardiac and skeletal muscle contractions, the properties of a family of Ca2+-binding proteins with 4 or 3 EF-hand motifs have been studied with desensitized skinned fiber preparations. Proteins with 4 EF hands (such as troponins C - TnCs) are dumb-bell shaped, those with 3 EF hands (parvalbumin) being ellipsoidal. The number of active sites varied between four and two. We find that the ability to anchor in the fiber is limited to proteins with 4 EF hands and, at least, two active Ca2+-binding sites, one each in the N- and C-termini. The results suggest that the dumb-bell shaped global structure is critical for the switching action in muscular contraction, and a trigger site in the N-terminus and a structural site in the C-terminus need to be active in order to regulate contractility.     

Isoelectric point mobility shift assay for rapid screening of charged and uncharged ligands bound to proteins

Three human proteins (hTAP1, hTAP2 and hTAP3) that are related to the yeast phosphatidylinositol/phosphatidylcholine transfer protein SEC14p were recently cloned in our laboratory. These proteins contain a relatively large hydrophobic pocket, the so called CRAL-TRIO domain, which is present also in other human proteins, such as CRALBP, alpha-TTP and MEG2. The CRAL-TRIO domains in these proteins bind ligands such as retinaldehyde, tocopherols and polyphosphoinositides, respectively. To screen for potential hTAPs ligands, we developed a semi-quantitative isoelectric point mobility shift assay (IPMS-assay) that allows assessing the binding of potential hydrophobic ligands to proteins. Purified proteins occupied with a charged ligand migrate differently on isoelectric focusing gels when compared with free protein. Competition of bound charged ligands with uncharged ones reverses the mobility shift, so that the relative affinities of the two ligands to the protein can be estimated.     

Analysis of the movements of helices in EF‐hands

We have developed a method to place an EF‐lobe in a coordinate system that recognizes the similarity of its two EF‐hand domains as well as their relationship by a pseudo‐two fold axis, z. The x‐axis connects the center of mass, calculated from α‐carbons of helices E1 and F1, with the center of mass of E2 and F2. The resulting coordinate system is intrinsic to each EF‐lobe and requires no comparison with other EF‐lobes. It has provided an intuitive and informative way to compare EF‐lobes and especially those changes associated with calcium and/or target binding. We analyzed the EF‐lobes of calmodulin and of other subfamilies with four EF‐hands. We have rationalized a complex pattern of changes of conformation associated with calcium coordination and effector binding as observed in different subfamilies of EF‐hand proteins. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Interaction of preS domains of hepatitis B virus with phospholipid vesicles

The role of preS domains of the hepatitis B virus (HBV) envelope proteins in the first steps of viral infection has been restricted to their implication in virus attachment to a putative hepatocyte receptor. In order to explore a fusion activity in these regions, we used recombinant preS domains to characterize their interaction with liposomes. Binding experiments carried out with NBD-labeled proteins indicated that preS were able to interact in a monomeric way with acidic phospholipid vesicles, being the partition coefficient similar to that described for peptides which can insert deeply into bilayers. Fluorescence depolarization of DPH-labeled vesicles confirmed the specificity for negative charged phospholipids. Upon interaction the proteins induced aggregation, lipid mixing and release of internal contents of acidic vesicles at both acid and neutral pH in a concentration-dependent manner. Taken together, all these data indicate that preS domains are able to insert into the hydrophobic core of the bilayer. Moreover, the insertion resulted in a protein conformational change which increased the helical content. Therefore all these results suggest that, besides their participation in the recognition of a cellular receptor, the preS domains could be involved in the fusion mechanism of HBV with the plasma membrane of target cells.     

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.     

Structure and dynamics of helix-0 of the N-BAR domain in lipid micelles and bilayers

Bin/Amphiphysin/Rvs-homology (BAR) domains generate and sense membrane curvature by binding the negatively charged membrane to their positively charged concave surfaces. N-BAR domains contain an N-terminal extension (helix-0) predicted to form an amphipathic helix upon membrane binding. We determined the NMR structure and nano-to-picosecond dynamics of helix-0 of the human Bin1/Amphiphysin II BAR domain in sodium dodecyl sulfate and dodecylphosphocholine micelles. Molecular dynamics simulations of this 34-amino acid peptide revealed electrostatic and hydrophobic interactions with the detergent molecules that induce helical structure formation from residues 8-10 toward the C-terminus. The orientation in the micelles was experimentally confirmed by backbone amide proton exchange. The simulation and the experiment indicated that the N-terminal region is disordered, and the peptide curves to adopted the micelle shape. Deletion of helix-0 reduced tubulation of liposomes by the BAR domain, whereas the helix-0 peptide itself was fusogenic. These findings support models for membrane curving by BAR domains in which helix-0 increases the binding affinity to the membrane and enhances curvature generation.     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

The crystal structure of plant-specific calcium-binding protein AtCBL2 in complex with the regulatory domain of AtCIPK14

Calcium signals mediate a multitude of plant responses to external stimuli. Calcineurin B-like (CBL) proteins and their target kinases, CBL-interacting protein kinases (CIPKs), represent important relays in plant calcium signaling. CBL interacts with CIPK through a conserved motif (NAF/FISL motif) within the C-terminal regulatory domain. To better understand the functional role of the CBL-CIPK system, we determined the crystal structure of AtCBL2 in complex with the regulatory domain of AtCIPK14 at 1.2 Å resolution. The NAF/FISL motif is inserted into a hydrophobic crevice within AtCBL2, accompanied by a large displacement of the helices and loop on the opposite side of the NAF/FISL motif from the C-terminal region, which shields the hydrophobic crevice in free form. Ca²⁺ are coordinated within four EF hands in AtCBL2 in bound form. This calcium coordination pattern differs from that in the structure of the SOS3-SOS2 complex previously reported. Structural comparison of the two structures shows that the recognition of CBL by CIPK is performed in a similar manner, but inherent interactions confer binding affinity and specificity.     

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.     

Nuclear magnetic resonance structure of calcium-binding protein 1 in a Ca(2+) -bound closed state: Implications for target recognition

Calcium‐binding protein 1 (CaBP1), a neuron‐specific member of the calmodulin (CaM) superfamily, regulates the Ca²⁺‐dependent activity of inositol 1,4,5‐triphosphate receptors (InsP3Rs) and various voltage‐gated Ca²⁺ channels. Here, we present the NMR structure of full‐length CaBP1 with Ca²⁺ bound at the first, third, and fourth EF‐hands. A total of 1250 nuclear Overhauser effect distance measurements and 70 residual dipolar coupling restraints define the overall main chain structure with a root‐mean‐squared deviation of 0.54 Å (N‐domain) and 0.48 Å (C‐domain). The first 18 residues from the N‐terminus in CaBP1 (located upstream of the first EF‐hand) are structurally disordered and solvent exposed. The Ca²⁺‐saturated CaBP1 structure contains two independent domains separated by a flexible central linker similar to that in calmodulin and troponin C. The N‐domain structure of CaBP1 contains two EF‐hands (EF1 and EF2), both in a closed conformation [interhelical angles = 129° (EF1) and 142° (EF2)]. The C‐domain contains EF3 and EF4 in the familiar Ca²⁺‐bound open conformation [interhelical angles = 105° (EF3) and 91° (EF4)]. Surprisingly, the N‐domain adopts the same closed conformation in the presence or absence of Ca²⁺ bound at EF1. The Ca²⁺‐bound closed conformation of EF1 is reminiscent of Ca²⁺‐bound EF‐hands in a closed conformation found in cardiac troponin C and calpain. We propose that the Ca²⁺‐bound closed conformation of EF1 in CaBP1 might undergo an induced‐fit opening only in the presence of a specific target protein, and thus may help explain the highly specialized target binding by CaBP1.     

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.     

Structure of apoptosis-linked protein ALG-2: insights into Ca2+-induced changes in penta-EF-hand proteins

BACKGROUND: The Ca2+ 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 Ca2+ binding sites. Like other PEF proteins, its N terminus contains a Gly/Pro-rich segment. Ca2+ binding is required for the interaction with the target protein, ALG-2 interacting protein 1 (AIP1). RESULTS: We present the 2.3 A resolution crystal structure of Ca2+-Ioaded des1-20ALG-2 (aa 21-191), which was obtained by limited proteolysis of recombinant ALG-2 with elastase. The molecule contains eight alpha helices that fold into five EF-hands, and, similar to other members of this protein family, the molecule forms dimers. Ca2+ ions bind to EF1, EF3, and, surprisingly, to EF5. In the related proteins calpain and grancalcin, the EF5 does not bind Ca2+ 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 Ca2+ and the peptide as compared to other Ca2+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 Ca2+ induces a conformational rearrangement in ALG-2, and that this mechanism is common to other PEF proteins.