Restraint-based comparative modeling was used for calculation and visualization of the H4-H5-loop of Na+/K+-ATPase from mouse brain (Mus musculus, adult male brain, α2-isoform) between the amino acid residues Cys336 and Arg758 in the E1 conformation The structure consists of two well separated parts. The N-domain is formed by a seven-stranded antiparallel β-sheet with two additional β-strands and five α-helices sandwiching it, the P-domain is composed of a typical Rossman fold. The ATP-binding site was found on the N-domain to be identical in both α2- and α1-isoforms. The phosphorylation Asp369 residue was found in the central part of the P-domain, located at the C-terminal end of the central β-sheet. The distance between the α-carbon of Phe475 at the ATP-binding site and the α-carbon of Asp369 at the phosphorylation site is 3.22 nm. A hydrogen bond between the oxygen atom of Asp369 and the nitrogen atom of Lys690 was clearly detected and assumed to play a key role in maintaining the proper structure of the physphorylaton site in E1 conformation., G. Tejral, L. Koláčná, A. Kotyk, E. Amler., and Obsahuje bibliografii
The distance between the ß-subunits of Na + /K + -ATPase isolated from pig dark red kidney medulla was determined by Forster energy transfer. First, oligosaccharides of the ß-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSFI were used as the donor and the acceptor, respectively, for Forster energy transfer studies to determine the localization of the ß-subunit in the native enzyme which is known to be formed as a tetramer (aß)2. It was found that the ß-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the ß-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor- acceptor pair CB-LY. The results also support the idea of a direct interaction of the ß-subunit with the extracellular part of the a-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or
time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys
501, Gly502 and Arg544, also Glu446, Phe475 and Gln 482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth
β-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is
directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.
Molecular modeling of the H4-H5-loop of the α2 isoform of Na+/K+-ATPase in the E1 and E2 conformations revealed that twisting of the nucleotide (N) domain toward the phosphorylation (P) domain is connected with the formation of a short π-helix between Asp369 and Thr375. This conformational change close to the hinge region between the N-domain and the P-domain could be an important event leading to a bending of the N-domain by 64.7° and to a shortening of the distance between the ATP binding site and the phosphorylation site (Asp369) by 1.22 nm from 3.22 nm to 2.00 nm. It is hypothesized that this shortening mechanism is involved in the Na+-dependent formation of the Asp369 phospho-intermediate as part of the overall Na+/K+-ATPase activity., G. Tejral ... [et al.]., and Obsahuje seznam literatury