Crystal structure of calcium binding protein-1 from Entamoeba histolytica: a novel arrangement of EF hand motifs

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.     

NMR derived solution structure of an EF-hand calcium-binding protein from Entamoeba Histolytica.

We present the three-dimensional (3D) solution structure of a calcium-binding protein from Entamoeba histolytica (EhCaBP), an etiologic agent of amoebiasis affecting millions worldwide. EhCaBP is a 14.7 kDa (134 residues) monomeric protein thought to play a role in the pathogenesis of amoebiasis. The 3D structure of Ca(2+)-bound EhCaBP has been derived using multidimensional nuclear magnetic resonance (NMR) spectroscopic techniques. The study reveals the presence of two globular domains connected by a flexible linker region spanning 8 amino acid residues. Each domain consists of a pair of helix-loop-helix motifs similar to the canonical EF-hand motif of calcium-binding proteins. EhCaBP binds to four Ca(2+) with high affinity (two in each domain), and it is structurally related to calmodulin (CaM) and troponin C (TnC) despite its low sequence homology ( approximately 29%) with these proteins. NMR-derived structures of EhCaBP converge within each domain with low RMSDs and angular order-parameters for backbone torsion angles close to 1.0. However, the presence of a highly flexible central linker region results in an ill-defined orientation of the two domains relative to one other. These findings are supported by backbone (15)N relaxation rate measurements and deuterium exchange studies, which reveal low structural order parameters for residues in the central linker region. Earlier, biochemical studies showed that EhCaBP is involved in a novel signal transduction mechanism, distinct from CaM. A possible reason for such a functional diversity is revealed by a detailed comparison of the 3D structure of EhCaBP with that of CaM and TnC. The studies indicate a more open C-terminal domain for EhCaBP with larger water exposed total hydrophobic surface area as compared to CaM and TnC. Further dissimilarities between the structures include the presence of two Gly residues (G63 and G67) in the central linker region of EhCaBP, which seem to impart it a greater flexibility compared to CaM and TnC and also play crucial role in its biological function. Thus, unlike in CaM and TnC, wherein the length and/or composition of the central linker have been found to be crucial for their function, in EhCaBP, both flexibility as well as amino acid composition is required for the function of the protein.     

Energetics of the native energy landscape of a two-domain calcium sensor protein: distinct folding features of the two domains

The protein folding energy landscape allows a thorough understanding of the protein folding problem which in turn helps in understanding various aspects of biological functions. Characterizing the cooperative unfolding units and the intermediates along the folding funnel of a protein is a challenging task. In this paper, we investigated the native energy landscape of EhCaBP, a calcium sensor, belonging to the same EF-hand superfamily as calmodulin. EhCaBP is a two-domain EF-hand protein consisting of two EF-hands in each domain and binding to four Ca2+ cations. Native-state hydrogen exchange (HX) was used to assess the folding features of the landscape and also to throw light on the structure-folding function paradigm of calcium sensor proteins. HX measurements under the EX2 regime provided the thermodynamic information about the protein folding events under native conditions. HX studies revealed that the unfolding of EhCaBP is not a two-state process. Instead, it proceeds through cooperative units. The C-terminal domain exhibits less denaturant dependence than the N-terminal domain, suggesting that the former is dominated by local fluctuations. It is interesting to note that the N- and C-terminal domains of EhCaBP have distinct folding features. In fact, these observed differences can regulate the domain-dependent target recognition of two-domain Ca2+ sensor proteins.     

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca²⁺ that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.     

Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein

Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways.     

Calcium-induced folding of a fragment of calmodulin composed of EF-hands 2 and 3

Calmodulin (CaM) is an EF-hand protein composed of two calcium (Ca(2+))-binding EF-hand motifs in its N-domain (EF-1 and EF-2) and two in its C-domain (EF-3 and EF-4). In this study, we examined the structure, dynamics, and Ca(2+)-binding properties of a fragment of CaM containing only EF-2 and EF-3 and the intervening linker sequence (CaM2/3). Based on NMR spectroscopic analyses, Ca(2+)-free CaM2/3 is predominantly unfolded, but upon binding Ca(2+), adopts a monomeric structure composed of two EF-hand motifs bridged by a short antiparallel beta-sheet. Despite having an "even-odd" pairing of EF-hands, the tertiary structure of CaM2/3 is similar to both the "odd-even" paired N- and C-domains of Ca(2+)-ligated CaM, with the conformationally flexible linker sequence adopting the role of an inter-EF-hand loop. However, unlike either CaM domain, CaM2/3 exhibits stepwise Ca(2+) binding with a K (d1) = 30 +/- 5 microM to EF-3, and a K (d2) > 1000 microM to EF-2. Binding of the first equivalent of Ca(2+) induces the cooperative folding of CaM2/3. In the case of native CaM, stacking interactions between four conserved aromatic residues help to hold the first and fourth helices of each EF-hand domain together, while the loop between EF-hands covalently tethers the second and third helices. In contrast, these aromatic residues lie along the second and third helices of CaM2/3, and thus are positioned adjacent to the loop between its "even-odd" paired EF-hands. This nonnative hydrophobic core packing may contribute to the weak Ca(2+) affinity exhibited by EF-2 in the context of CaM2/3.     

Crystal-structure and biochemical characterization of recombinant human calcyphosine delineates a novel EF-hand-containing protein family

Calcyphosine is an EF-hand protein involved in both Ca^2 +-phosphatidylinositol and cyclic AMP signal cascades, as well as in other cellular functions. The crystal structure of Ca^2 +-loaded calcyphosine was determined up to 2.65 Å resolution and reveals a protein containing two pairs of Ca^2 +-binding EF-hand motifs. Calcyphosine shares a highly similar overall topology with calmodulin. However, there are striking differences between EF-hand 4, both N-terminal and C-terminal regions, and interdomain linkers. The C-terminal domain of calcyphosine possesses a large hydrophobic pocket in the presence of calcium ions that might be implicated in ligand binding, while its N-terminal hydrophobic pocket is almost shielded by an additional terminal helix. Calcyphosine is largely monomeric, regardless of the presence of Ca^2 +. Differences in structure, oligomeric state in the presence and in the absence of Ca^2 +, a highly conserved sequence with low similarity to other proteins, and phylogeny define a new EF-hand-containing family of calcyphosine proteins that extends from arthropods to humans.     

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.     

Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.     

The conformational plasticity of calmodulin upon calcium complexation gives a model of its interaction with the oedema factor of Bacillus anthracis

We analyzed the conformational plasticity of calmodulin (CaM) when it is bound to the oedema factor (EF) of Bacillus anthracis and its response to calcium complexation with molecular dynamics (MD) simulations. The EF-CaM complex was simulated during 15 ns for three different levels of calcium bound to CaM. They were respectively no calcium ion (EF-(Apo-CaM)), two calcium ions bound to the C-terminal domain of CaM (EF-(2Ca-CaM)), and four calcium ions bound to CaM (EF-(4Ca-CaM)). Calculations were performed using AMBER package. The analysis of the MD simulations illustrates how CaM forces EF in an open conformation to form the adenylyl cyclase enzymatic site, especially with the two calcium form of CaM, best suited to fit the open conformation of EF. By contrast, CaM encounters bending and unwinding of its flexible interlinker in EF-(Apo-CaM) and EF-(4Ca-CaM). Calcium binding to one domain of CaM affects the other one, showing a transmission of information along the protein structure. The analysis of the CaM domains conformation along the simulations brings an atomistic and dynamic explanation for the instability of these complexes. Indeed the EF-hand helices of the N-terminal domain tend to open upon calcium binding (EF-(4Ca-CaM)), although the domain is locked by EF. By contrast, the C-terminal domain is strongly locked in the open conformation by EF, and the removal of calcium induces a collapse of EF catalytic site (EF-(Apo-CaM)).     

A 1.3-A structure of zinc-bound N-terminal domain of calmodulin elucidates potential early ion-binding step

Calmodulin (CaM) is a 16.8-kDa calcium-binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix-loop-helix calcium-binding motif. CaM is composed of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3-Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single-wavelength anomalous diffraction phasing of a selenomethionyl N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-calmodulin (apo-CaM), despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a cross-linked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggest that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either stepwise conformational transition or flexible, dynamic energetic state sampling-based transition.     

Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.     

Functional interaction of yeast elongation factor 3 with yeast ribosomes

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.     

Structural characterization of a novel Ca2+-binding protein from Entamoeba histolytica: structural basis for the observed functional differences with its isoform

A novel Ca²⁺-binding protein (EhCaBP2) was identified from the protozoan parasite Entamoeba histolytica. EhCaBP2 has 79% sequence identity with calcium-binding protein EhCaBP1. The 3D structure of EhCaBP2 was determined using multidimensional nuclear magnetic resonance spectroscopic techniques. The study reveals that the protein consists of two globular domains connected by a short flexible linker region of four residues. On comparison of the 3D structure and dynamics of EhCaBP2 with those of EhCaBP1, it is found that they vary significantly in their N-terminal domains and interdomain linker. Immunofluorescence localization experiments revealed that EhCaBP1 and EhCaBP2 may not carry out similar functions, as their cellular distribution patterns are not the same. The functional differences between the two isoforms are explained on the basis of results obtained from the structural studies. The structural variation in the interdomain linker region and the formation of functionally important hydrophobic clefts in different regions of EhCaBP1 and EhCaBP2 provide interesting insights into the differences in the functionality of these two isoforms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. S. M. Mustafi and R. B. Mutalik equally contributed to this work.     

Domain stability and metal-induced folding of calcium- and integrin-binding protein 1

It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-hand proteins such as calmodulin or troponin C. In this work we investigate the structure and stability of the four EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presence and absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structure and flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimented with steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1 proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II (F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structure similar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain. The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partially unfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stable structure. The data reveal significant communication between the N- and C-lobes of CIB1, and show that transient intermediate conformations are formed along the unfolding pathway for each form of the protein. Collectively the data demonstrate that the communication between the paired EF-hand domains as well as between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C, which might be important for the unique function of CIB1 in numerous biological processes.     

The N-terminal domain of MDM2 resembles calmodulin and its relatives.

It is shown here that the N-terminal domain of MDM2, which is not thought to bind calcium ions, otherwise bears a striking resemblance to a cluster of four EF-hand modules like those found in the calmodulin family. There are similarities in module arrangement, supersecondary structure and the main-chain to main-chain hydrogen-bonding pattern, especially in the vicinity of the short antiparallel beta-sheet, the two strands of which lie between the two E and F helices of tandem modules. Some conserved amino acid residues are identified that are associated with short side-chain to main-chain hydrogen-bonded motifs. Also, both types of domain bind a short, functionally important hydrophobic alpha-helix from another protein in a cavity between the two pairs of EF-hand, or EF-hand-like, modules.     

A novel extracellular EF-hand protein involved in the shell formation of pearl oyster

Mollusk shell formation is a complicated and highly controlled calcium metabolism process. Previous studies revealed that several EF-hand calcium-binding proteins actively participate in the regulation of shell mineralization. In this study, we cloned a full-length cDNA encoding a novel extracellular EF-hand calcium-binding protein (named EFCBP) from the pearl oyster, Pinctada fucata, according to the EF-hand motifs of calmodulin. Although it shares high similarity with the calmodulin family in its EF-hand signatures, EFCBP just has two EF-hand motifs and belongs to a new separate group from the other EF-hand proteins according to a phylogenetic analysis. EFCBP is specifically expressed in shell mineralization-related tissues, viz. the mantle, the gill, and the hemocytes. Moreover, its expression responds quickly only to the shell damage, but not to the damage of other tissues and the infection of the lipopolysaccharides from Escherichia coli. These results suggest that EFCBP might be an important regulator of shell formation. This finding may help better understand the functions of EF-hand proteins on the regulation of mollusk shell formation.     

Apo-calmodulin binds with its C-terminal domain to the N-methyl-D-aspartate receptor NR1 C0 region

Calmodulin (CaM) is the major Ca2+ sensor in eukaryotic cells. It consists of four EF-hand Ca2+ binding motifs, two in its N-terminal domain and two in its C-terminal domain. Through a negative feedback loop, CaM inhibits Ca2+ influx through N-methyl-D-aspartate-type glutamate receptors in neurons by binding to the C0 region in the cytosolic tail of the NR1 subunit. Ca2+ -depleted (apo)CaM is pre-associated with a variety of ion channels for fast and effective regulation of channel activities upon Ca2+ influx. Using the NR1 C0 region for fluorescence and circular dichroism spectroscopy studies we found that not only Ca2+ -saturated CaM but also apoCaM bound to NR1 C0. In vitro interaction assays showed that apoCaM also binds specifically to full-length NR1 solubilized from rat brain and to the complete C terminus of the NR1 splice form that contains the C0 plus C2' domain. The Ca2+ -independent interaction of CaM was also observed with the isolated C-but not N-terminal fragment of calmodulin in the independent spectroscopic assays. Fluorescence polarization studies indicated that apoCaM associated via its C-terminal domain with NR1 C0 in an extended conformation and collapsed to adopt a more compact conformation of faster rotational mobility in its complex with NR1 C0 upon addition of Ca2+. Our results indicate that apoCaM is associated with NR1 and that the complex of CaM bound to NR1 C0 undergoes a dramatic conformational change when Ca2+ binds to CaM.     

Structure of the N-terminal calcium sensor domain of centrin reveals the biochemical basis for domain-specific function

Centrin is an essential component of microtubule-organizing centers in organisms ranging from algae and yeast to humans. It is an EF-hand calcium-binding protein with homology to calmodulin but distinct calcium binding properties. In a previously proposed model, the C-terminal domain of centrin serves as a constitutive anchor to target proteins, and the N-terminal domain serves as the sensor of calcium signals. The three-dimensional structure of the N-terminal domain of Chlamydomonas rheinhardtii centrin has been determined in the presence of calcium by solution NMR spectroscopy. The domain is found to occupy an open conformation typical of EF-hand calcium sensors. Comparison of the N- and C-terminal domains of centrin reveals a structural and biochemical basis for the domain specificity of interactions with its cellular targets and the distinct nature of centrin relative to other EF-hand proteins. An NMR titration of the centrin N-terminal domain with a fragment of the known centrin target Sfi1 reveals binding of the peptide to a discrete site on the protein, which supports the proposal that the N-terminal domain serves as a calcium sensor in centrin.