Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM.

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Disruption of cagA, the apoprotein gene of chromoprotein antibiotic C-1027, eliminates holo-antibiotic production, but not the cytotoxic chromophore.

C-1027 is a chromoprotein of the nine-membered enediyne antitumour antibiotic family, comprising apoprotein to stabilize and transport the enediyne chromophore. The disruption of apoprotein gene cagA within the C-1027 biosynthetic gene cluster abolished C-1027 holo-antibiotic production detected by an antibacterial assay, as well as the expression of the apoprotein and C-1027 chromophore extracted following protein precipitation of the culture supernatant. Complementation of the cagA-disrupted mutant AKO with the intact cagA gene restored C-1027 production, suggesting that cagA is indispensable for holo-antibiotic production. Overexpression of cagA in the wild-type strain resulted in a significant increase in C-1027 production as expected. Surprisingly, electrospray ionization (ESI)-MS and ESI-MS/MS analyses suggested that the AKO mutant still produced the C-1027 enediyne chromophore [m/z=844 (M+H)(+)] and its aromatized product [m/z=846 (M+H)(+)]. Consistent with this, the results from gene expression analysis using real-time reverse transcriptase-PCR showed that transcripts of the positive regulator sgcR3 and the structural genes sgcA1, sgcC4, sgcD6 and sgcE were readily detected in the AKO mutant as well as in the wild-type and the complementation strain. These results provided, for the first time, evidence suggesting that the apoprotein of C-1027 is not essential in the self-resistance mechanism for the enediyne chromophore.

Structure-induced strain determining the internal cyclization site in the yeast cobI5 autocatalytic intron: theory and experimental tests.

The terminal intron of the apocytochrome b gene of yeast is endowed with autocatalytic potential in vitro and has been also shown to be capable of internal circularization at the linkage 236-237 from the 5' extremity. The aim of this work is to identify the secondary interaction responsible for shaping and activating the internal cyclization site (ICS). This is done by simulating the sequential folding of an abnormally large fragment of 237 nucleotides which starts at the 5' cleavage site and contains the internal guide sequence (IGS) starting 220 nucleotides into the intron. The resulting portion of the overall structure features the conserved interaction P1 and is compatible with the complete consensus secondary structure for this intron. The structural motif which emerges from sequential folding and is responsible for shaping the ICS is confirmed by analyzing the localization of structure-induced strain in the RNA backbone. It is shown that after the conserved stem P1 has been dismantled, the highest strain in a phosphodiester linkage is localized internally precisely at the experimentally determined cyclization site. Moreover, it is shown that our dynamical model of folding is compatible with the actual reaction pathways. Thus, internal cyclization becomes feasible only after conserved interaction P1 has been dismantled. This last event, in turn, might take place as a consequence of either of the following events: (i) 5' cleavage caused by nucleophilic attack by the GTP-cosubstrate or (ii) excision of the 3' exon after prior formation of P10. This last event, in turn, requires dismantling helix P1. Moreover, event (ii) is necessary for internal circularization since it habilitates the 3' terminal guanosine as a nucleophilic agent.