Characterization of the ATP-binding domain of the sarco(endo)plasmic reticulum Ca2+-ATPase: probing nucleotide binding by multidimensional NMR

ABSTRACT: The skeletal muscle sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a) mediates muscle relaxation by pumping Ca 2+ from the cytosol to the ER/SR lumen. In efforts aimed at understanding the structural basis for the conformational changes accompanying the reaction cycle catalyzed by SERCA1a, we have studied the ATP-binding domain of SERCA1a in both nucleotide-bound and -free forms by NMR. Limited proteolysis analyses guided us to express a 28 kDa stably folded fragment containing the nucleotide-binding domain of SERCA1a spanning residues Thr357-Leu600. ATP binding activity was demonstrated for this fragment by a FITC competition assay. A nearly complete backbone resonance assignment of this 28 kDa ATP-binding fragment, in both the AMP-PNP-bound and -free forms, was obtained by means of heteronuclear multidimensional NMR techniques. NMR titration experiments with AMP-PNP revealed a confined nucleotide-binding site which coincides with a cytoplasmic pocket region identified in the crystal structure of apo-SERCA1a. These results are consistent with previous site-directed mutagenesis studies of SERCA1a

Effects of a Glucan from the Edible Mushroom (Pleurotus florida) as an Immunostimulant in Farmed Indian Major Carp (Catla catla)

The purpose of this study was to determine the effects of a dietary mushroom glucan on the non- specific immune responses and disease resistance of the Indian major carp, Catla catla. Glucan was extracted from farm-raised edible mushrooms (Pleurotus florida). The glucan was fed to test animals for two weeks at 0.5 g or 1 g/kg feed. Afterward, the fish received the control diet for another four weeks. Non-specific immune responses were measured at the end of the two-week experimental feeding (week 0), two weeks later (week 2), and four weeks later (week 4). Control and test fish were challenged by intraperitoneal injection of the fish pathogenic bacterium, Aeromonas hydrophila, seven days after the two-week experimental feeding period. Mortality was observed and the relative percent survival was calculated. In the 0.5 g treatment, significant enhancement (p0.05), but lysozyme activity was raised at weeks 2 and 4 in the 0.5 g treatment and at week 2 in the 1 g treatment. Both con- centrations significantly enhanced relative percent survival

Angucycline C5 Glycosides: Regio- and Stereocontrolled Synthesis and Cytotoxicity

This study discloses a general and convergent route for the regio- and stereospecific construction of the C5 glycosyl angucycline framework of mayamycin. <i>C</i>-Glycosidation, dearomatization, and Hauser annulation are the key steps. The synthetic analogues show cytotoxicity against different human cancer cell lines with IC₅₀ values between 16.4 and 1.2 μM

Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB

The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II

Genetic characterization of the Entamoeba moshkovskii population based on different potential genetic markers

Entamoeba moshkovskii, according to recent studies, appears to exert a more significant impact on diarrhoeal infections than previously believed. The efficient identification and genetic characterization of E. moshkovskii isolates from endemic areas worldwide are crucial for understanding the impact of parasite genomes on amoebic infections. In this study, we employed a multilocus sequence typing system to characterize E. moshkovskii isolates, with the aim of assessing the role of genetic variation in the pathogenic potential of E. moshkovskii. We incorporated 3 potential genetic markers: KERP1, a protein rich in lysine and glutamic acid; amoebapore C (apc) and chitinase. Sequencing was attempted for all target loci in 68 positive E. moshkovskii samples, and successfully sequenced a total of 33 samples for all 3 loci. The analysis revealed 17 distinct genotypes, labelled M1–M17, across the tested samples when combining all loci. Notably, genotype M1 demonstrated a statistically significant association with diarrhoeal incidence within E. moshkovskii infection (P = 0.0394). This suggests that M1 may represent a pathogenic strain with the highest potential for causing diarrhoeal symptoms. Additionally, we have identified a few single-nucleotide polymorphisms in the studied loci that can be utilized as genetic markers for recognizing the most potentially pathogenic E. moshkovskii isolates. In our genetic diversity study, the apc locus demonstrated the highest Hd value and π value, indicating its pivotal role in reflecting the evolutionary history and adaptation of the E. moshkovskii population. Furthermore, analyses of linkage disequilibrium and recombination within the E. moshkovskii population suggested that the apc locus could play a crucial role in determining the virulence of E. moshkovskii

Abbreviations used: IP 3 R, inositol 1,4

⁎ Cytoplasmic Ca 2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R), which functions as a Ca 2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP 3 R have been reported; those of the IP 3 -binding core (IP 3 R CORE ) with bound IP 3 , and the suppressor domain. This study examines the structural effects of ligand binding on an IP 3 R construct, designated IP 3 R N , that contains both the IP 3 -binding core and the suppressor domain. Our circular dichroism results reveal that the IP 3 -bound and IP 3 -free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP 3 has a large effect on the stability of IP 3 R CORE , it has little effect on IP 3 R N , indicating that the suppressor domain is critical to the stability of IP 3 R N . The NMR data for IP 3 R N provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP 3 -bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP 3 R N undergoes a change in average conformation in response to IP 3 binding and the presence of Ca 2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP 3 R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP 3 binding drives the conformational equilibrium toward more compact structures, while the presence of Ca 2+ drives it to a more extended set