Structural basis for the observed differential magnetic anisotropic tensorial values in calcium binding proteins

Lanthanide ions (Ln(3+)), which have ionic radii similar to those of Ca(2+), can displace the latter in a calcium binding protein, without affecting its tertiary structure. The paramagnetic Ln(3+) possesses large anisotropic magnetic susceptibilities and produce pseudocontact shifts (PCSs), which have r(-3) dependence. The PCS can be seen for spins as far as 45 A from the paramagnetic ion. They aid in structure refinement of proteins by providing long-range distance constraints. Besides, they can be used to determine the interdomain orientation in multidomain proteins. This is particularly important in the context of a calcium binding protein from Entamoeba histolytica (EhCaBP), which consists of two globular domains connected by a flexible linker region containing 8 residues. As a first step to obtain the interdomain orientation in EhCaBP, a suite of 2D and 3D heteronuclear experiments were recorded on EhCaBP by displacing calcium with Ce(3+), Ho(3+), Er(3+), Tm(3+), Dy(3+), and Yb(3+) ions in separate experiments, and the PCS of (1)H(N) and (15)N spins were measured. Such data have been used in the refinement of the individual domain structures of the protein in parallel with the calculation of the respective magnetic anisotropy tensorial values, which differ substantially (2.1-2.8 times) from what is found in other Ca(2+) binding loops. This study provides a structural basis for such variations in the magnetic anisotropy tensorial values.     

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.     

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.     

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants. The results of this paper have been granted US Patent No. 6,791,009.     

Crystal structure of the tandem phosphatase domains of RPTP LAR.

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR revealed that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) conferred a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution.     

Crystal Structure of CBD2 from the Drosophila Na/Ca Exchanger: Diversity of Ca Regulation and Its Alternative Splicing Modification

Na⁺/Ca²⁺ exchangers (NCXs) promote the extrusion of intracellular Ca²⁺ to terminate numerous Ca²⁺-mediated signaling processes. Ca²⁺ interaction at two Ca²⁺ binding domains (CBDs; CBD1 and CBD2) is important for tight regulation of the exchange activity. Diverse Ca²⁺ regulatory properties have been reported with several NCX isoforms; whether the regulatory diversity of NCXs is related to structural differences of the pair of CBDs is presently unknown. Here, we reported the crystal structure of CBD2 from the Drosophila melanogaster exchanger CALX1.1. We show that the CALX1.1-CBD2 is an immunoglobulin-like structure, similar to mammalian NCX1-CBD2, but the predicted Ca²⁺ interaction region of CALX1.1-CBD2 is arranged in a manner that precludes Ca²⁺ binding. The carboxylate residues that coordinate two Ca²⁺ in the NCX1-CBD1 structure are neutralized by two Lys residues in CALX1.1-CBD2. This structural observation was further confirmed by isothermal titration calorimetry. The CALX1.1-CBD2 structure also clearly shows the alternative splicing region forming two adjacent helices perpendicular to CBD2. Our results provide structural evidence that the diversity of Ca²⁺ regulatory properties of NCX proteins can be achieved by (1) local structure rearrangement of Ca²⁺ binding site to change Ca²⁺ binding properties of CBD2 and (2) alternative splicing variation altering the protein domain-domain conformation to modulate the Ca²⁺ regulatory behavior.     

Shape and Flexibility in the Titin 11-Domain Super-Repeat

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of ∼ 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 μm long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the ‘large super-repeat’. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 μm long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerised when bound to the thick filament.     

Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn α-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and ¹⁵N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.     

Determination of the Individual Roles of the Linker Residues in the Interdomain Motions of Calmodulin Using NMR Chemical Shifts

Many protein molecules are formed by two or more domains whose structures and dynamics are closely related to their biological functions. It is thus important to develop methods to determine the structural properties of these multidomain proteins. Here, we characterize the interdomain motions in the calcium-bound state of calmodulin (Ca^2 +-CaM) using NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations. We find that the conformational fluctuations of the interdomain linker, which are largely responsible for the overall interdomain motions of CaM, can be well described by exploiting the information provided by chemical shifts. We thus identify 10 residues in the interdomain linker region that change their conformations upon substrate binding. Five of these residues (Met76, Lys77, Thr79, Asp80 and Ser81) are highly flexible and cover the range of conformations observed in the substrate-bound state, while the remaining five (Arg74, Lys75, Asp78, Glu82 and Glu83) are much more rigid and do not populate conformations typical of the substrate-bound form. The ensemble of conformations representing the Ca^2 +-CaM state obtained in this study is in good agreement with residual dipolar coupling, paramagnetic resonance enhancement, small-angle X-ray scattering and fluorescence resonance energy transfer measurements, which were not used as restraints in the calculations. These results provide initial evidence that chemical shifts can be used to characterize the conformational fluctuations of multidomain proteins.     

The calcium binding protein EhCaBP6 is a microtubular‐end binding protein in Entamoeba histolytica

The genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF‐hand domains EFI and EFIII; it can bind Ca²⁺ in vitro and undergoes conformational transition on binding Ca²⁺ suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress. Unlike other Ca²⁺ binding proteins that have been studied in E. histolytica, EhCaBP6 is found at microtubule ends and at the intercellular bridge with the microtubules during cytokinesis. Furthermore, increased expression of EhCaBP6 was correlated with a significant increase in the number of microtubular structures suggesting that this protein may regulate chromosome segregation and cytokinesis in E. histolytica.     

Light-induced structural changes of LOV domain-containing polypeptides from Arabidopsis phototropin 1 and 2 studied by small-angle X-ray scattering

Phototropin is a blue-light receptor of plants and comprises two light-receptive domains, LOV1 and LOV2, Ser/Thr kinase domain and one linker region connecting the LOV2 and the kinase domains. The LOV2 domain is thought to regulate predominantly the light-dependent autophosphorylation of the kinase domain, leading to cellular signaling cascades. In this study, we constructed recombinant LOV1, LOV2, and LOV2-linker polypeptides from phototropin 1 and phototropin 2 of Arabidopsis thaliana and studied their quaternary structures and light-dependent conformational changes by small-angle X-ray scattering. The molecular weights of the polypeptides determined from scattering intensities demonstrated the dimeric associations of LOV1 polypeptides of both isoforms. In contrast, while LOV2 and LOV2-linker polypeptides of phototropin 1 were homodimers, corresponding polypeptides of phototropin 2 existed as monomeric forms. Under blue-light irradiation, the LOV2-linker polypeptide of phototropin 1 displayed small but definite changes of the scattering profile. Through simulation of low-resolution molecular structures, the changes were likely explained as structural changes of the linker region and/or a movement of the region relative to the LOV2 domain. Light-induced profile changes were not observed in the Cys(512)Ala mutated LOV2-linker polypeptide of phototropin 1 losing the phototransformation capability. Thus, it was indicated that the photoreaction in the LOV2 domain probably caused the structural changes in the LOV2-linker polypeptide of phototropin 1. On the basis of the results, the interdomain interactions in phototropin are discussed.     

Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel

The voltage-gated H⁺ channel (Hv) is a H⁺-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.     

Structural basis for sequential displacement of Ca(2+) by Yb(3+) in a protozoan EF-hand calcium binding protein

We have studied the displacement of Ca(2+)by the trivalent lanthanide ions (Yb(3+)) in a protozoan (Entamoeba histolytica) Ca(2+)-binding protein (EhCaBP), by NMR and thermodynamics. We have demonstrated, for the first time, how one can use in a combined fashion the utility of NMR and thermodynamics to have an insight to the relative binding specificities/affinity between Ca(2+) and Yb(3+). As revealed by the titration experiments, Yb(3+) displaces Ca(2+) from the four metal binding sites present in EhCaBP in a sequential manner. The study provides a structural origin for such a sequential Ca(2+) displacement by Yb(3+) in EhCaBP.     

Molecular Dynamics Study of the Structure,Flexibility and Dynamics of Thermostable L1 Lipase at High Temperatures

Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn²⁺-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn²⁺-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Crystal structure of the FERM domain of focal adhesion kinase

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.     

Interdomain linkage in the polymeric hemoglobin molecule of Artemia

Artemia has evolved the longest known concatenation of hemoglobin domains, the subunit containing nine domains and the subunit having a similar size. Translation of the cDNA sequence of the subunit reveals eight regions of inter-domain polypeptide linking together the nine heme-binding domains, together with partially analogous sequences preceding the first domain and following the last. Analysis of the structural possibilities of the linker sequences suggests how the domains may be organized in the subunit.The interdomain linker sequences were 14%–64% identical (62%–91% similar by Dayhoff substitution matrix) and approximately 14 residues in length including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu-. The linker composition resembled that of the 11 amino acid pre-A leader sequence of Petromyzon marinus (lamprey) hemoglobin V, the structure of which is known. Prediction of structure from the Artemia linker sequences indicated a nonhelical, turn-associated linker which could be modeled to the Petromyzon leader. Measurements confirmed that such a structure could support the packing of nine Artemia domains into a polymeric subunit of annular shape, two of which subunits (which can be similar or dissimilar) comprise the physiological molecule.The position of interdomain introns and the character of a variable residue early in the linker are compatible with the nine-domain polymer having evolved through gene duplication reflected in globin domain fusion incorporating an extension specifically of the N-terminus. The multiplication of an original single-domain globin gene to give the present nine is estimated from sequence differences, allowing for multiple mutations at individual sites, to have occurred in a period at least 500–700 million years ago.Correspondence to: C.N.A. Trotman 1444     

Differences in stability and calcium sensitivity of the Ig domains in titin's N2A region

Titin is a large filamentous protein that spans half a sarcomere, from Z‐disk to M‐line. The N2A region within the titin molecule exists between the proximal immunoglobulin (Ig) region and the PEVK region and protein–protein interactions involving this region are required for normal muscle function. The N2A region consists of four Ig domains (I80–I83) with a 105 amino acid linker region between I80 and I81 that has a helical nature. Using chemical stability measurements, we show that predicted differences between the adjacent Ig domains (I81–I83) correlate with experimentally determined differences in chemical stability and refolding kinetics. Our work further shows that I83 has the lowest ΔG_unfolding, which is increased in the presence of calcium (pCa 4.3), indicating that Ca²⁺ plays a role in stabilizing this immunoglobulin domain. The characteristics of N2A's three Ig domains provide insight into the stability of the binding sites for proteins that interact with the N2A region. This work also provides insights into how Ca²⁺ might influence binding events involving N2A.     

Chimeric Fc receptors identify ligand binding regions in human glycoprotein VI

Glycoprotein (GP) VI, a key receptor for collagen-induced platelet activation, recently emerged as a major target for developing new antithrombotics. However, little is known about its functional domains, which is a disadvantage for the rational development of antagonists. Our aim was to identify the structures determining GPVI specificity. GPVI presents homologies with members of the Ig superfamily (in particular with FcalphaRI) whose extracellular parts present two domains, D1 and D2 linked by a hinge interdomain. To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis. Proteins were tested for their binding to ligands and antibodies specific for GPVI and FcalphaRI. We demonstrate for the first time that D2 plays a specific and significant role in GPVI binding to collagen and that the hinge interdomain is critical for the binding to convulxin. Furthermore, binding to CRP requires elements of D1 and of the linker motif. Our results indicate that GPVI is unique amongst the receptors of its family as it uses different structural domains to interact with several agonists and provide evidence that different sites on GPVI constitute targets to develop antagonists of GPVI.     

HSPA1A conformational mutants reveal a conserved structural unit in Hsp70 proteins

BackgroundThe Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown.MethodsHere, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays.ResultsAnalysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein.ConclusionsThese findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery.General significanceMutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.