Interactions of anti-sigma factor antagonists of Mycobacterium tuberculosis in the yeast two-hybrid system

Anti-sigma factor antagonists (anti-anti-sigma factors) play critical roles in regulating the expression of alternative sigma factors in response to specific stress signals. The Clusters of Orthologous Groups (COG) database has identified the existence of six genes, Rv0516c, Rv1364c, Rv1365c, Rv1904, Rv2638 and Rv3687c (grouped under the cluster COG1366), encoding potential anti-sigma factor antagonists in Mycobacterium tuberculosis. These molecules are speculated to regulate the expression of sigma factor SigF of M. tuberculosis in response to stress signals. Since signaling occurs via physical interactions of proteins (protein-protein interaction), we investigated whether the anti-sigma factor antagonists of M. tuberculosis interact with anti-sigma factor RsbW (Rv3287c) or the sigma factor SigF (Rv3286c) in the yeast two-hybrid system. The results revealed that most of the anti-sigma factor antagonists interact with either RsbW or SigF or both. In addition, some anti-sigma factor antagonists also displayed limited interactions between themselves. These interactions suggest that they possibly transduce some signals to SigF and between themselves.     

Role of Mg block of the inward rectifier K current in cardiac repolarization reserve: A quantitative simulation

Different K⁺ currents serve as “repolarization reserve” or a redundant repolarizing mechanism that protects against excessive prolongation of the cardiac action potential and therefore arrhythmia. Impairment of the inward rectifier K⁺ current (I_K1) has been implicated in the pathogenesis of cardiac arrhythmias. The characteristics of I_K1 reflect the kinetics of channel block by intracellular cations, primarily spermine (a polyamine) and Mg²⁺, whose cellular levels may vary under various pathological conditions. However, the relevance of endogenous I_K1 blockers to the repolarization reserve is still not fully understood in detail. Here we used a mathematical model of a cardiac ventricular myocyte which quantitatively reproduces the dynamics of I_K1 block to examine the effects of the intracellular spermine and Mg²⁺ concentrations, through modifying I_K1, on the action potential repolarization. Our simulation indicated that an I_K1 transient caused by relief of Mg²⁺ block flows during early phase 3. Increases in the intracellular spermine/Mg²⁺ concentration, or decreases in the intracellular Mg²⁺ concentration, to levels outside their normal ranges prolonged action potential duration by decreasing the I_K1 transient. Moreover, reducing both the rapidly activating delayed rectifier current (I_Kr) and the I_K1 transient caused a marked retardation of repolarization and early afterdepolarization because they overlap in the voltage range at which they flow. Our results indicate that the I_K1 transient caused by relief of Mg²⁺ block is an important repolarizing current, especially when I_Kr is reduced, and that abnormal intracellular free spermine/Mg²⁺ concentrations may be a missing risk factor for malignant arrhythmias in I_Kr-related acquired (drug-induced) and congenital long QT syndromes.     

Growth regulation, reverse transformation, and adaptability of 3T3 cells in decreased Mg2+ concentration

A nontransformed and a spontaneously transformed clone of BALB/c 3T3 cells were compared for their capacity to multiply in decreased concentrations of Mg²⁺. Cells of the nontransformed clone were flat, formed regularly patterned, nonoverlapping arrays, required high serum concentration for multiplication, had a low saturation density, and did not make colonies in agar. Cells of the transformed clone were slender and spiky, formed random, overlapping arrays, multiplied in low serum concentrations, and had no fixed saturation density, and 20-30% of them formed colonies in agar. The saturation density of the nontransformed clone was decreased in a growth-limiting supply of Mg²⁺ in proportion to the reduction in initial rate of multiplication. At very low Mg²⁺ concentrations, saturation occurred when less than half of the surface of the dish was covered with cells. The transformed cells did not reach a stable saturation density in low Mg²⁺ concentrations, but their growth rate did slow down when they became crowded, and a transient saturation density was reached at the lowest Mg²⁺ concentrations that allowed multiplication. Limiting the supply of Mg²⁺ caused the transformed cells to flatten and to assume a regularly patterned, non-overlapping relationship to one another, resembling that of the nontransformed cells. This also occurred in BALB/c 3T3 cells transformed by infection with Moloney mouse sarcoma virus. After 1 week in low concentrations of Mg²⁺, the nontransformed cells began to multiply and to incorporate [³H]thymidine at a rapid rate. The transformed cells did so also and, in addition, reverted to their transformed appearance. The intracellular content of Mg²⁺ was not significantly decreased when the extracellular concentration was decreased to 1/50th. The results suggest that: (a) limited contact among cells already multiplying at a reduced rate is sufficient to halt further multiplication; (b) a very small decrease in intracellular Mg²⁺ content or in membrane-associated Mg²⁺ causes transformed cells to assume aspects of the appearance and behavior of nontransformed cells (i.e., Mg²⁺-regulated reactions may be involved in determining the transformed phenotype); and (c) cells multiplying at a slow rate in low concentrations of Mg²⁺ begin to multiply faster after about 1 week, due either to an adaptation of the cells or to a change in the cellular microenvironment.     

Mammalian MagT1 and TUSC3 are required for cellular magnesium uptake and vertebrate embryonic development

Magnesium (Mg²⁺) is the second most abundant cation in cells, yet relatively few mechanisms have been identified that regulate cellular levels of this ion. The most clearly identified Mg²⁺ transporters are in bacteria and yeast. Here, we use a yeast complementary screen to identify two mammalian genes, MagT1 and TUSC3, as major mechanisms of Mg²⁺ influx. MagT1 is universally expressed in all human tissues and its expression level is up-regulated in low extracellular Mg²⁺. Knockdown of either MagT1 or TUSC3 protein significantly lowers the total and free intracellular Mg²⁺ concentrations in mammalian cell lines. Morpholino knockdown of MagT1 and TUSC3 protein expression in zebrafish embryos results in early developmental arrest; excess Mg²⁺ or supplementation with mammalian mRNAs can rescue the effects. We conclude that MagT1 and TUSC3 are indispensable members of the vertebrate plasma membrane Mg²⁺ transport system.     

Structural insights into the mechanisms of Mg2+ uptake,transport, and gating by CorA

Despite the importance of Mg²⁺ for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg²⁺ transport. However, the key questions—such as, the ion selectivity, the transport pathway, and the gating mechanism—have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg²⁺ during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg²⁺ to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.     

Interplay of Mg2+, ADP,and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg²⁺ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using ³¹P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg²⁺ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg²⁺ in the mitochondrial matrix, where [Mg²⁺] is tenfold higher. In contrast, owing to a much higher affinity for Mg²⁺, ATP is mostly complexed by Mg²⁺ in both compartments. Mg²⁺ starvation used to alter cytosolic and mitochondrial [Mg²⁺] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg²⁺ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.In heterotrophic and well-oxygenated plant cells, ATP is regenerated from ADP principally by glycolysis and mitochondrial oxidative phosphorylation. Surprisingly, although ATP synthesis mechanisms have been deciphered for decades, whether cell respiration is controlled by [ATP]/[ADP] or [ATP]/[ADP][Pi] ratios (1, 2), by the adenylate energy charge ([ATP + 0.5 ADP]/[ATP + ADP + AMP]) (3, 4), and/or by the concentration of ATP or ADP in the cytosol (5, 6) remains a matter of debate. To our knowledge, the determining factor for controlling cell respiration in response to the energy demand has not yet been unambiguously characterized.MgATP is the substrate of numerous phosphorylating enzymes and the principal energy source of the cell. Indeed, any increase in metabolic activity increases the rate of MgATP use and, consequently, the rate of ADP and magnesium release, and vice versa. In normoxia, the MgATP concentration should be essentially balanced by the ADP phosphorylation catalyzed by mitochondrial ATP synthase, thereby adjusting oxidative phosphorylation to cell ATP needs. The ADP/ATP carrier (AAC) of the inner mitochondrial membrane, which exchanges free nucleotides, and adenylate kinase (EC 2.7.4.3), which interconverts MgADP and free ADP with MgATP and free AMP in the presence of Mg²⁺ (7), participate in this regulation (reviewed in ref. 8). Clearly, to better understand the interplay of free and Mg-complexed ADP and ATP in the regulation of cell respiration it is necessary to know their concentrations, as well as the concentration of Mg²⁺ in the cytosol and mitochondrial matrix.Nucleotides can be measured using ³¹P-NMR spectroscopy both in vitro, from cell extracts, and in vivo, in perfused material. After 1 h of data accumulation time, detection thresholds are approximately 20 nmol in vitro and 50 nmol in vivo (9). Various techniques for measuring intracellular [Mg²⁺] and free/Mg-complexed nucleotides have been proposed (10–12), but none allows measurement in different intracellular compartments. In vivo ³¹P-NMR spectroscopy offers this possibility, because the chemical shift (δ) of the γ- and β-phosphorus resonances of ATP and the β-phosphorus resonance of ADP depend on pH and [Mg²⁺] (13). We adapted this noninvasive technique to the simultaneous in vivo measurement of cytosolic and mitochondrial Mg²⁺ and free/Mg-complexed nucleotides concentrations in culture cells.We used homogenous cells cultivated on liquid nutrient media (NM) so as to narrow resonance peaks on in vivo NMR spectra, thus improving the signal-to-noise ratios and the accuracy of chemical shift measurements and limiting peak overlaps. In addition, the heterotrophic sycamore (Acer pseudoplatanus L.) cells of cambial origin used in this study contain no large chloroplasts, but only small plastids (14, 15) with low amounts of nucleotides (16), thus permitting more precise measurement of the cytosolic and mitochondrial nucleotide pools.To modify nucleotide concentrations without using inhibitors that may interfere with mitochondrial functioning, we varied the cell culture media: standard, adenine-supplied, Pi-starved, and Mg-starved. In this paper, we refer to cytoplasm as the cell compartment exterior to the vacuole and cytosol as the cell compartment exterior to the vacuole and the organelles bounded by a double membrane (mitochondria and plastids).The aim of the present study was to determine the role of ADP, ATP, and Mg²⁺ concentrations in the in vivo control of mitochondrial respiration. We show that the balance between cytosolic and mitochondrial free ADP, depending on the concentration of Mg²⁺ in the cytosol and matrix, mediates this regulation.     

Structural asymmetry in the magnesium channel CorA points to sequential allosteric regulation

Magnesium ions (Mg²⁺) are essential for life, but the mechanisms regulating their transport into and out of cells remain poorly understood. The CorA-Mrs2-Alr1 superfamily of Mg²⁺ channels represents the most prevalent group of proteins enabling Mg²⁺ ions to cross membranes. Thermotoga maritima CorA (TmCorA) is the only member of this protein family whose complete 3D fold is known. Here, we report the crystal structure of a mutant in the presence and absence of divalent ions and compare it with previous divalent ion-bound TmCorA structures. With Mg²⁺ present, this structure shows binding of a hydrated Mg²⁺ ion to the periplasmic Gly-Met-Asn (GMN) motif, revealing clues of ion selectivity in this unique channel family. In the absence of Mg²⁺, TmCorA displays an unexpected asymmetric conformation caused by radial and lateral tilts of protomers that leads to bending of the central, pore-lining helix. Molecular dynamics simulations support these movements, including a bell-like deflection. Mass spectrometric analysis confirms that major proteolytic cleavage occurs within a region that is selectively exposed by such a bell-like bending motion. Our results point to a sequential allosteric model of regulation, where intracellular Mg²⁺ binding locks TmCorA in a symmetric, transport-incompetent conformation and loss of intracellular Mg²⁺ causes an asymmetric, potentially influx-competent conformation of the channel.     

Rifamycins do not function by allosteric modulation of binding of Mg2+ to the RNA polymerase active center

Rifamycin antibacterial agents inhibit bacterial RNA polymerase (RNAP) by binding to a site adjacent to the RNAP active center and preventing synthesis of RNA products >2–3 nt in length. Recently, Artsimovitch et al. [(2005) Cell 122:351–363] proposed that rifamycins function by allosteric modulation of binding of Mg²⁺ to the RNAP active center and presented three lines of biochemical evidence consistent with this proposal. Here, we show that rifamycins do not affect the affinity of binding of Mg²⁺ to the RNAP active center, and we reassess the three lines of biochemical evidence, obtaining results not supportive of the proposal. We conclude that rifamycins do not function by allosteric modulation of binding of Mg²⁺ to the RNAP active center.     

Solubilization of a calcium dependent adenosine triphosphatase from rat heart sarcolemma

The rat heart sarcolemmal ATPase activity was found to be dependent on the presence of Ca²⁺ (v_max = 37 μmol Pi/mg protein/h and K_a = 0.59 mm) or Mg²⁺ ions (v_max = 28 μmol Pi/mg protein/h and K_a = 0.87 mm). The incubation of sarcolemmal membrane with trypsin stimulated the Ca²⁺ dependent ATPase activity without affecting the Mg²⁺ dependent ATPase activity. The increase in Ca²⁺ dependent ATPase activity was associated with a decrease in the K_a value from 0.59 to 0.45 mm and an increase in v_max from 37 to 69 μmol Pi/mg protein/h. In membrane preparations treated with detergents, trypsin decreased the Mg²⁺ dependent ATPase activity whereas the Ca²⁺ dependent ATPase activity was increased. Trypsin treatment of sarcolemma released proteins in the supernatant that showed ATP hydrolysis in the presence of Ca²⁺ but not Mg²⁺; while the residual membrane showed an ATPase dependent on Ca²⁺ (v_max = 67 μmol Pi/mg protein/h and K_a = 0.58 mm) and Mg²⁺ (v_max = 58 μmol Pi/mg protein/h and K_a = 0.87 mm). The proteins released in the supernatant were subjected to column chromatography on sepharose-6B, and DEAE cellulose and a Ca²⁺-dependent ATPase activity (v_max = 200 μmol Pi/mg protein/h and K_a = 0.25 mm) was recovered as a distinct peak. These results indicate solubilization of a Ca²⁺ dependent ATPase which, unlike the enzyme present in heart sarcolemma, showed negligible activation by Mg²⁺.