Isolation and characterization of grancalcin, a novel 28 kDa EF-hand calcium-binding protein from human neutrophils.
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ABSTRACT:
SUBMITTER: Teahan CG
PROVIDER: S-EPMC1132932 | biostudies-other | 1992 Sep
REPOSITORIES: biostudies-other
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Project description:A novel 22.8 kDa of Opisthorchis viverrini (Ov) calcium-binding EF-hand protein (Ov CaBP) was identified and isolated from an immunoscreening of the adult stage Ov cDNA library by using a human cholangiocarcinoma (CCA) serum. This protein was related to other calcium-binding proteins and conserved among the trematodes. Ov CaBP shared 98% amino acid identity to 22.8 kDa of Clonorchis sinensis CaBP and both were classified as a new group of CaBP EF-hand protein by multiple sequence alignment and phylogenetic tree analysis. The open reading frame of Ov CaBP was 585 bp which encoded for 194 amino acids. The N-terminal part is composed of two calcium-binding EF-hand motifs whereas the C-terminal part contains a dynein light chain motif (DLC). In addition, transcription analysis by RT-PCR revealed that it was constitutively transcribed in all stages, including metacercariae, juvenile, and adult. Furthermore, recombinant Ov CaBP protein (rOv CaBP) was expressed as a soluble protein and antibody generated against this rOv CaBP protein was capable of detecting Ov CaBP in the Ov somatic extracts but not in Ov ES products. This anti-rOv CaBP serum was also used to localize Ov CaBP in Ov infected hamster's liver sections which the distribution of Ov CaBP was located in gut epithelium, miracidia in eggs and slightly in parenchyma. Moreover, rOv CaBP protein showed a calcium-binding property in non-denaturing gel mobility shift assay.
| S-EPMC3728565 | biostudies-literature
Project description:Four isoforms of calcium binding proteins containing 2 EF hand motifs and a dynein light chain-like domain in the human liver fluke Opisthorchis viverrini, namely OvCaBP1, 2, 3, and 4, were characterized. They had molecular weights of 22.7, 21.6, 23.7, and 22.5 kDa, respectively and showed 37.2-42.1% sequence identity to CaBP22.8 of O. viverrini. All were detected in 2- and 4-week-old immature and mature parasites. Additionally, OvCaBP4 was found in newly excysted juveniles. Polyclonal antibodies against each isoform were generated to detect the native proteins in parasite extracts by Western blot analysis. All OvCaBPs were detected in soluble and insoluble crude worm extracts and in the excretory-secretory product, at approximate sizes of 21-23 kDa. The ion-binding properties of the proteins were analyzed by mobility shift assays with the divalent cations Ca2+, Mg2+, Zn2+, and Cu2+. All OvCaBPs showed mobility shifts with Ca2+ and Zn2+. OvCaBP1 showed also positive results with Mg2+ and Cu2+. As tegumental proteins, OvCaBP1, 2, and 3 are interesting drug targets for the treatment of opisthorchiasis.
| S-EPMC5858670 | biostudies-literature
Project description:The EF hand, a helix-loop-helix structure, is one of the most common motifs found in animal genomes, and EF-hand Ca(2+)-binding proteins (EFCaBPs) are widely distributed throughout the cell. However, researchers remain confounded by a lack of understanding of how peptide sequences code for specific functions and by uncertainty about the molecular mechanisms that enable EFCaBPs to distinguish among many diverse cellular targets. Such knowledge could define the roles of EFCaBPs in health and disease and ultimately enable control or even design of Ca(2+)-dependent functions in medicine and biotechnology. In this Account, we describe our structural and biochemical research designed to understand the sequence-to-function relationship in EFCaBPs. The first structural goal was to define conformational changes induced by binding Ca(2+), and our group and others established that solution NMR spectroscopy is well suited for this task. We pinpointed residues critical to the differences in Ca(2+) response of calbindin D(9k) and calmodulin (CaM), homologous EFCaBPs from different functional classes, by using direct structure determination with site-directed mutagenesis and protein engineering. Structure combined with biochemistry provided the foundation for identifying the fundamental mechanism of cooperativity in the binding of Ca(2+) ions: this cooperativity provides EFCaBPs with the ability to detect the relatively small changes in concentration that constitute Ca(2+) signals. Using calbindin D(9k) as a model system, studies of the structure and fast time scale dynamics of each of the four ion binding states in a typical EF-hand domain provided direct evidence that site-site communication lowers the free energy cost of reorganization for binding the second ion. Our work has also extended models of how EFCaBPs interact with their cellular targets. We determined the unique dimeric architecture of S100 proteins, a specialized subfamily of EFCaBPs found exclusively in vertebrates. We described the implications for how these proteins transduce signals and went on to characterize interactions with peptide fragments of important cellular targets. Studies of the CaM homolog centrin revealed novel characteristics of its binding of Ca(2+) and its interaction with its cellular target Kar1. These results provided clear examples of how subtle differences in sequence fine-tune EFCaBPs to interact with their specific targets. The structural approach stands at a critical crossroad, shifting in emphasis from descriptive structural biochemistry to integrated biology and medicine. We present our dual-molecular-switch model for Ca(2+) regulation of gating functions of voltage-gated sodium channels in which both CaM and an intrinsic EF-hand domain serve as coupled Ca(2+) sensors. A second example involves novel EFCaBP extracellular function, that is, the role of S100A8/S100A9 heterodimer in the innate immune response to bacterial pathogens. A mechanism for the antimicrobial activity of S100A8/S100A9 was discovered. We describe interactions of S100A8/S100A9 and S100B with the cell surface receptor for advanced glycation end products. Biochemical and structural studies are now uncovering the mechanisms by which EFCaBPs work and are helping to define their biological activities, while simultaneously expanding knowledge of the roles of these proteins in normal cellular physiology and the pathology of disease.
| S-EPMC3059389 | biostudies-literature
Project description:The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an "EF-hand-like" calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.
| S-EPMC4022735 | biostudies-literature
Project description:Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca(2+) in EF-hand proteins, which is crucial to function.
| S-EPMC2576379 | biostudies-literature
Project description:Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr2+ interference with Ca2+ binding to proteins of the EF-hand family, we studied Sr2+/Ca2+ interaction with a canonical EF-hand protein, α-parvalbumin (α-PA). Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA ('CD' and 'EF' sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M-1 and 4 × 109 M-1 for Ca2+, and 2 × 107 M-1 and 2 × 106 M-1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M-1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO- groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.
| S-EPMC8392015 | biostudies-literature
Project description:Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K1=7.1+/-0.4, log K2=4.7+/-0.6, log K3=3.6+/-0.7 and log K4=4.6+/-0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 microM. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K(d) was determined to be 1.2x10(-7) M in the presence of Ca2+ and 1.5x10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.
| S-EPMC1698678 | biostudies-literature
Project description:Tescalcin (TESC) is an EF-hand calcium binding protein that is differentially expressed in several tissues, however it is not reported that the expression and functional roles of TESC in colorectal cancer. Levels of messenger RNA (mRNA) and protein expression of TESC in colorectal cancer tissues were assessed using RT-PCR, real time PCR, immunohistochemistry, and clinicopathologic analyses. Quantitative analysis of TESC levels in serum specimens was performed using sandwich ELISA. Colorectal cancer cells transfected with TESC small interfering RNA and short hairpin RNA were examined in cell proliferation assays, phospho-MAPK array, and mouse xenograft models. Here we demonstrated that TESC is overexpressed in colorectal cancer (CRC), but was not expressed in normal mucosa and premalignant dysplastic lesions. Furthermore, serum TESC levels were elevated in patients with CRC. Knockdown of TESC inhibited the Akt-dependent NF-κB pathway and decreased cell survival in vitro. Depletion of TESC reduced tumor growth in a CRC xenograft model. Thus, TESC is a potential diagnostic marker and oncotarget in colorectal cancer.
| S-EPMC4039152 | biostudies-literature
Project description:Members of the parvalbumin (PV) family of calcium (Ca2+) binding proteins (CBPs) share a relatively high level of sequence similarity. However, their Ca2+ affinities and selectivities against competing ions like Mg2+ can widely vary. We conducted molecular dynamics simulations of several α-parvalbumin (αPV) constructs with micromolar to nanomolar Ca2+ affinities to identify structural and dynamic features that contribute to their binding of ions. Specifically, we examined a D94S/G98E construct with a lower Ca2+ affinity (≈-18 kcal/mol) relative to the wild type (WT) (≈-22 kcal/mol) and an S55D/E59D variant with enhanced affinity (≈-24 kcal/mol). Additionally, we also examined the binding of Mg2+ to these isoforms, which is much weaker than Ca2+. We used mean spherical approximation (MSA) theory to evaluate ion binding thermodynamics within the proteins' EF-hand domains to account for the impact of ions' finite sizes and the surrounding electrolyte composition. While the MSA scores differentiated Mg2+ from Ca2+, they did not indicate that Ca2+ binding affinities at the binding loop differed between the PV isoforms. Instead, molecular mechanics generalized Born surface area (MM/GBSA) approximation energies, which we used to quantify the thermodynamic cost of structural rearrangement of the proteins upon binding ions, indicated that S55D/E59D αPV favored Ca2+ binding by -20 kcal/mol relative to WT versus 30 kcal/mol for D94S/G98E αPV. Meanwhile, Mg2+ binding was favored for the S55D/E59D αPV and D94S/G98E αPV variants by -18.32 and -1.65 kcal/mol, respectively. These energies implicate significant contributions to ion binding beyond oxygen coordination at the binding loop, which stemmed from changes in α-helicity, β-sheet character, and hydrogen bonding. Hence, Ca2+ affinity and selectivity against Mg2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions elsewhere. Our findings broaden our understanding of the molecular bases governing αPV ion binding that are likely shared by members of the broad family of CBPs.
| S-EPMC8848088 | biostudies-literature
Project description:Leucine Zipper EF-hand containing transmembrane protein-1 (LETM1) is an inner mitochondrial membrane protein that mediates mitochondrial calcium (Ca2+ )/proton exchange. The matrix residing carboxyl (C)-terminal domain contains a sequence identifiable EF-hand motif (EF1) that is highly conserved among orthologues. Deletion of EF1 abrogates LETM1 mediated mitochondrial Ca2+ flux, highlighting the requirement of EF1 for LETM1 function. To understand the mechanistic role of this EF-hand in LETM1 function, we characterized the biophysical properties of EF1 in isolation. Our data show that EF1 exhibits α-helical secondary structure that is augmented in the presence of Ca2+ . Unexpectedly, EF1 features a weak (~mM), but specific, apparent Ca2+ -binding affinity, consistent with the canonical Ca2+ coordination geometry, suggested by our solution NMR. The low affinity is, at least in part, due to an Asp at position 12 of the binding loop, where mutation to Glu increases the affinity by ~4-fold. Further, the binding affinity is sensitive to pH changes within the physiological range experienced by mitochondria. Remarkably, EF1 unfolds at high and low temperatures. Despite these unique EF-hand properties, Ca2+ binding increases the exposure of hydrophobic regions, typical of EF-hands; however, this Ca2+ -induced conformational change shifts EF1 from a monomer to higher order oligomers. Finally, we showed that a second, putative EF-hand within LETM1 is unreactive to Ca2+ either in isolation or tandem with EF1. Collectively, our data reveal that EF1 is structurally and biophysically responsive to pH, Ca2+ and temperature, suggesting a role as a multipartite environmental sensor within LETM1.
| S-EPMC7980503 | biostudies-literature