Molecular Mechanisms of Enzyme Activation by Monovalent Cations.
Ontology highlight
ABSTRACT:
SUBMITTER: Gohara DW
PROVIDER: S-EPMC5076497 | biostudies-literature | 2016 Sep
REPOSITORIES: biostudies-literature
Publications
Molecular Mechanisms of Enzyme Activation by Monovalent Cations.
Gohara David W DW Di Cera Enrico E
The Journal of biological chemistry 20160726 40
Regulation of enzymes through metal ion complexation is widespread in biology and underscores a physiological need for stability and high catalytic activity that likely predated proteins in the RNA world. In addition to divalent metals such as Ca<sup>2+</sup>, Mg<sup>2+</sup>, and Zn<sup>2+</sup>, monovalent cations often function as efficient and selective promoters of catalysis. Advances in structural biology unravel a rich repertoire of molecular mechanisms for enzyme activation by Na<sup>+</ ...[more]
Similar Datasets
Project description:Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na+ as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M+ ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[13C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li+ to Cs+, PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. PFL-AE is thus a type I M+-activated enzyme whose M+ controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.
| S-EPMC5579537 | biostudies-literature
Project description:The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.
| S-EPMC7278361 | biostudies-literature
Project description:The composition of the ion atmosphere surrounding nucleic acids affects their folding, condensation and binding to other molecules. It is thus of fundamental importance to gain predictive insight into the formation of the ion atmosphere and thermodynamic consequences when varying ionic conditions. An early step toward this goal is to benchmark computational models against quantitative experimental measurements. Herein, we test the ability of the three dimensional reference interaction site model (3D-RISM) to reproduce preferential interaction parameters determined from ion counting (IC) experiments for mixed alkali chlorides and dsDNA. Calculations agree well with experiment with slight deviations for salt concentrations >200 mM and capture the observed trend where the extent of cation accumulation around the DNA varies inversely with its ionic size. Ion distributions indicate that the smaller, more competitive cations accumulate to a greater extent near the phosphoryl groups, penetrating deeper into the grooves. In accord with experiment, calculated IC profiles do not vary with sequence, although the predicted ion distributions in the grooves are sequence and ion size dependent. Calculations on other nucleic acid conformations predict that the variation in linear charge density has a minor effect on the extent of cation competition.
| S-EPMC4787805 | biostudies-literature
Project description:Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A.U and G.C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (<15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G.C helices were unaffected by changing salt concentration, A.U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U.C.U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.
| S-EPMC2528546 | biostudies-literature
Project description:Acinetobacter baumannii is well adapted to the hospital environment, where infections caused by this organism are associated with significant morbidity and mortality. Genetic determinants of antimicrobial resistance have been described extensively, yet the mechanisms by which A. baumannii regulates antibiotic resistance have not been defined. We sought to identify signals encountered within the hospital setting or human host that alter the resistance phenotype of A. baumannii. In this regard, we have identified NaCl as being an important signal that induces significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin upon the culturing of A. baumannii cells in physiological NaCl concentrations. Proteomic analyses of A. baumannii culture supernatants revealed the release of outer membrane proteins in high NaCl, including two porins (CarO and a 33- to 36-kDa protein) whose loss or inactivation is associated with antibiotic resistance. To determine if NaCl affected expression at the transcriptional level, the transcriptional response to NaCl was determined by microarray analyses. These analyses highlighted 18 genes encoding putative efflux transporters that are significantly upregulated in response to NaCl. Consistent with this, the effect of NaCl on the tolerance to levofloxacin and amikacin was significantly reduced upon the treatment of A. baumannii with an efflux pump inhibitor. The effect of physiological concentrations of NaCl on colistin resistance was conserved in a panel of multidrug-resistant isolates of A. baumannii, underscoring the clinical significance of these observations. Taken together, these data demonstrate that A. baumannii sets in motion a global regulatory cascade in response to physiological NaCl concentrations, resulting in broad-spectrum tolerance to antibiotics.
| S-EPMC2825970 | biostudies-literature
Project description:Four new crystal structures of the ATPase domain of the GyrB subunit of Escherichia coli DNA gyrase have been determined. One of these, solved in the presence of K(+), is the highest resolution structure reported so far for this domain and, in conjunction with the three other structures, reveals new insights into the function of this domain. Evidence is provided for the existence of two monovalent cation-binding sites: site 1, which preferentially binds a K(+) ion that interacts directly with the α-phosphate of ATP, and site 2, which preferentially binds an Na(+) ion and the functional significance of which is not clear. The crystallographic data are corroborated by ATPase data, and the structures are compared with those of homologues to investigate the broader conservation of these sites.
| S-EPMC4388272 | biostudies-literature
Project description:We have obtained a 1.55-Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni under conditions that permit detailed observations of Na(+) ion binding in the ribozyme's active site. At least two such Na(+) ions are observed. The first Na(+) ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical with that previously observed for divalent cations. A second Na(+) ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with that of previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na(+), but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na(+) directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active-site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest-resolution ribozyme structure in the Protein Data Bank.
| S-EPMC3795999 | biostudies-literature
Project description:RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion-phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties.
| S-EPMC6800359 | biostudies-literature
Project description:While multimodal (MM) chromatography is a promising approach for purifying proteins, the lack of a fundamental understanding of how ion-ligand interactions govern selectivity limits its use in the biopharmaceutical industry. This study uses molecular dynamics simulations to study the interactions between simple monovalent cations and two commonly used structurally similar multimodal chromatography ligands, the Capto ligand and Nuvia cPrime, immobilized on the surface. On the Capto ligand surface, ion presence and type play a key role in modulating the formation of phenyl rings and carboxylate clusters. The flexible linkage attaching the Capto ligand to the self-assembled monolayer (SAM) surface allowed multiple ligands to form interactions with the small cations, while large cations interacted less strongly, following the order Li+ > Na+ > K+ > Cs+. Thus, smaller cations resulted in greater ordering on the surface and lower ion diffusivities, while larger cations resulted in less ordering and higher ion diffusivities, following the order Li+ < Na+ < K+ < Cs+. In contrast, due to the rigid attachment of Nuvia cPrime to the SAM surfaces, the cations bound less strongly and had a much smaller effect on ligand clustering or ordering. Additionally, ions in the presence of the Nuvia cPrime surface had generally greater diffusivities than those in the presence of the Capto ligand. Overall, the interaction of cations with the multimodal ligands can lead to unique configurations on the SAM that likely contribute to differential behavior in biological separations.
| S-EPMC10993413 | biostudies-literature
Project description:The opening of the permeability transition pore (PTP) in mitochondria is a key event in the initiation of cell death in various pathologic states, including ischemia/reperfusion. The activation of K+ transport into mitochondria protects cells from ischemia/reperfusion. However, the role of K+ transport in PTP regulation is unclear. Here, we studied the role of K+ and other monovalent cations in the regulation of the PTP opening in an in vitro model. The registration of the PTP opening, membrane potential, Ca2+-retention capacity, matrix pH, and K+ transport was performed using standard spectral and electrode techniques. We found that the presence of all cations tested in the medium (K+, Na+, choline+, and Li+) strongly stimulated the PTP opening compared with sucrose. Several possible reasons for this were examined: the effect of ionic strength, the influx of cations through selective and non-selective channels and exchangers, the suppression of Ca2+/H+ exchange, and the influx of anions. The data obtained indicate that the mechanism of PTP stimulation by cations includes the suppression of K+/H+ exchange and acidification of the matrix, which facilitates the influx of phosphate. Thus, the K+/H+ exchanger and the phosphate carrier together with selective K+ channels compose a PTP regulatory triad, which might operate in vivo.
| S-EPMC10252971 | biostudies-literature