An alignment of upstream regions of anaerobically induced genes in Staphylococcus aureus revealed the presence of an inverted repeat, corresponding to Rex binding sites in Streptomyces coelicolor. Gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator Rex of S. aureus binds to this inverted repeat. The binding sequence – TTGTGAAW4TTCACAA – is highly conserved in S. aureus. Rex binding to this sequence leads to the repression of genes located downstream. The binding activity of Rex is enhanced by NAD+ while NADH, which competes with NAD+ for Rex binding, decreases the activity of Rex. The impact of Rex on global protein synthesis and on the activity of fermentation pathways under aerobic and anaerobic conditions was analysed by using a rex-deficient strain. A direct regulatory effect of Rex on the expression of pathways that lead to anaerobic NAD+ regeneration, such as lactate, formate and ethanol formation, nitrate respiration, and ATP synthesis, is verified. Rex can be considered a central regulator of anaerobic metabolism in S. aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon.

SummaryThe transcription factor Rex has been implicated in regulation of the expression of genes important for fermentative growth and for growth under conditions of low oxygen tension in several Gram-positive bacteria. Rex senses the redox poise of the cell through changes in the NADH/NAD + ratio. The crystal structures of two essentially identical Rex proteins, from Thermus aquaticus and T. thermophilus, have previously been determined in complex with NADH. Here we present the crystal structure of the Rex protein from Bacillus subtilis, as well as extensive studies of its affinity for nucleotides and DNA, using surface plasmon resonance, isothermal titration calorimetry and electrophoretic mobility shift assays. We show that Rex has a very high affinity for NADH but that its affinity for NAD + is 20 000 times lower. However, the NAD + affinity is increased by a factor of 30 upon DNA binding, suggesting that there is a positive allosteric coupling between DNA binding and NAD + binding. The crystal structures of two pseudoapo forms (from crystals soaked with NADH and cocrystallized with ATP) show a very different conformation from the previously determined Rex:NADH complexes, in which the N-terminal domains are splayed away from the dimer core. A mechanism is proposed whereby conformational changes in a C-terminal domain-swapped helix mediate the transition from a flexible DNA binding form to a locked NADH-bound form incapable of binding DNA.

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.

Calmodulin (CaM) is the central mediator of intracellular Ca2+ signalling in cardiomyocytes, where it conveys the intricate Ca 2+ transients to the proteins controlling cardiac contraction. We recently linked two separate mutations in CaM (N53I and N97S) to dominantly inherited catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmic disorder in which exercise or acute emotion can lead to syncope and sudden cardiac death. Given the ubiquitous presence of CaM in all eukaryote cells, it is particular intriguing that carriers of either mutation show no additional symptoms. Here, we investigated the effects of the CaM CPVT mutations in a zebrafish animal model. Three-day-old embryos injected with either CaM mRNA showed no detectable pathologies or developmental abnormalities. However, embryos injected with CPVT CaM mRNA displayed increased heart rate compared to wild-type CaM mRNA under b-adrenergic stimulation, demonstrating a conserved dominant cardiac specific effect between zebrafish and human carriers of these mutations. Motivated by the highly similar physiological phenotypes, we compared the effects of the N53I and N97S mutations on the biophysical and functional properties of CaM. Surprisingly, the mutations have opposing effects on CaM C-lobe Ca 2+ binding affinity and kinetics, and changes to the CaM N-lobe Ca 2+ binding are minor and specific to the N53I mutation. Furthermore, both mutations induce differential perturbations to structure and stability towards unfolding. Our results suggest different molecular disease mechanisms for the CPVT (N53I and N97S mutations) and strongly support that cardiac contraction is the physiological process most sensitive to CaM integrity.

Calmodulin is an essential regulator of intracellular processes in response to extracellular stimuli mediated by a rise in Ca 2؉ ion concentration. To profile protein-protein interactions of calmodulin in human brain, we probed a high content human protein array with fluorophore-labeled calmodulin in the presence of Ca