Characterization of a Protease-resistant Domain of the Cytosolic Portion of Sarcoplasmic Reticulum Ca2+-ATPase

Skilandat

Metal Ions in Life Sciences

et al. 2012

Cadmium(II), commonly classified as a relatively soft metal ion, prefers indeed aromaticnitrogen sites (e.g., N7 of purines) over oxygen sites (like sugar-hydroxyl groups). However, matters are not that simple, though it is true that the affinity of Cd(2+) towards ribose-hydroxyl groups is very small; yet, a correct orientation brought about by a suitable primary binding site and a reduced solvent polarity, as it is expected to occur in a folded nucleic acid, may facilitate metal ion-hydroxyl group binding very effectively. Cd(2+) prefers the guanine(N7) over the adenine(N7), mainly because of the steric hindrance of the (C6)NH(2) group in the adenine residue. This Cd(2+)-(N7) interaction in a guanine moiety leads to a significant acidification of the (N1)H meaning that the deprotonation reaction occurs now in the physiological pH range. N3 of the cytosine residue, together with the neighboring (C2)O, is also a remarkable Cd(2+) binding site, though replacement of (C2)O by (C2)S enhances the affinity towards Cd(2+) dramatically, giving in addition rise to the deprotonation of the (C4)NH(2) group. The phosphodiester bridge is only a weak binding site but the affinity increases further from the mono-to the di-and the triphosphate. The same also holds for the corresponding nucleotides. Complex stability of the pyrimidine-nucleotides is solely determined by the coordination tendency of the phosphate group(s), whereas in the case of purine-nucleotides macrochelate formation takes place by the interaction of the phosphate-coordinated Cd(2+) with N7. The extents of the formation degrees of these chelates are summarized and the effect of a non-bridging sulfur atom in a thiophosphate group (versus a normal phosphate group) is considered. Mixed ligand complexes containing a nucleotide and a further mono-or bidentate ligand are covered and it is concluded that in these species N7 is released from the coordination sphere of Cd(2+). In the case that the other ligand contains an aromatic residue (e.g., 2,2'-bipyridine or the indole ring of tryptophanate) intramolecular stack formation takes place. With buffers like Tris or Bistris mixed ligand complexes are formed. Cd(2+) coordination to dinucleotides and to dinucleoside monophosphates provides some insights regarding the interaction between Cd(2+) and nucleic acids. Cd(2+) binding to oligonucleotides follows the principles of coordination to its units. The available crystal studies reveal that N7 of purines is the prominent binding site followed by phosphate oxygens and other heteroatoms in nucleic acids. Due to its high thiophilicity, Cd(2+) is regularly used in so-called thiorescue experiments, which lead to the identification of a direct involvement of divalent metal ions in ribozyme catalysis.

supporting

confidence: 84%

Section: Ternary Cadmium(ii) Complexes Containing Atp 4-and a Buffer mentioning

confidence: 99%

Complex Formation of Cadmium with Sugar Residues, Nucleobases, Phosphates, Nucleotides, and Nucleic Acids

Skilandat

Metal Ions in Life Sciences

et al. 2012

Journal of Biological Chemistry

“…For this purpose, labeling with FITC was performed by incubating control SR or PK-SR membranes at about 2 mg/ml protein with substoichiometric amounts of FITC (either 4 or 2 nmol of FITC/mg of protein) for 40 min at 20°C and pH 8, followed by return to neutral pH and storage on ice (32). We found previously (33) that in the isolated water-soluble p29/30 fragments formed after extensive ATPase proteolysis with PK, FITC specifically reacted with Lys 515 , as in intact ATPase. We therefore anticipated that in the p83C-28N complex resulting from milder proteolysis, FITC would also react with Lys 515 , again in the same manner as with intact ATPase.…”

mentioning

confidence: 73%

Calcium Transport by Sarcoplasmic Reticulum Ca2+-ATPase

Møller

Lenoir

Marchand

et al. 2002

Self Cite

2؉and a protecting non-phosphorylated ligand (e.g. adenosine 5-(␤,␥-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE 243 sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca 2؉ and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADPsensitive phosphoenzyme, Ca 2 E1P, with occluded Ca 2؉ . The ability of Ca 2 E1P to become converted to the Ca 2؉ -free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca 2؉ . PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca 2؉ . It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca 2؉ ions.

“…Thus also the formation of nucleic acid ternary complexes with buffers and metal ions, like they have been observed for nucleotides [57,307,308,311,321] and in human serum albumin [322], is a possibility that should be kept in mind when studying the interactions of metal ions and nucleic acids.…”

Section: Effect Of Buffersmentioning

confidence: 93%

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

Pechlaner

Metal Ions in Life Sciences

2011

Metal ions are inextricably involved with nucleic acids due to their polyanionic nature. In order to understand the structure and function of RNAs and DNAs, one needs to have detailed pictures on the structural, thermodynamic, and kinetic properties of metal ion interactions with these biomacromolecules. In this review we first compile the physicochemical properties of metal ions found and used in combination with nucleic acids in solution. The main part then describes the various methods developed over the past decades to investigate metal ion binding by nucleic acids in solution. This includes for example hydrolytic and radical cleavage experiments, mutational approaches, as well as kinetic isotope effects. In addition, spectroscopic techniques like EPR, lanthanide(III) luminescence, IR and Raman as well as various NMR methods are summarized. Aside from gaining knowledge about the thermodynamic properties on the metal ion-nucleic acid interactions, especially NMR can be used to extract information on the kinetics of ligand exchange rates of the metal ions applied. The final section deals with the influence of anions, buffers, and the solvent permittivity on the binding equilibria between metal ions and nucleic acids. Little is known on some of these aspects, but it is clear that these three factors have a large influence on the interaction between metal ions and nucleic acids.