1Laboratory of High Performance Computing, Institute of Physical Biology USB and Institute of Landscape Ecology AS CR, University of South Bohemia, Zámek 136, CZ-373 33 Nové Hrady, Czech Republic, email: email@example.com
2Institute of Physics, Charles University, Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic
3Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12840 Prague 2, Czech Republic
4Institute of Physiology of AS CR, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic
5Institute of Biophysics, Faculty of Medicine, Charles University, V Úvalu 84, CZ-15000 Prague 5, Czech Republic
The NMR solved structure of Na+/K+-ATPase N-domain  represents the first breakthrough in solving the structure of the ATP-binding domain with a high resolution method. However, Hilge et al.  marginalized the role of protein molecular modeling done prior to their NMR-study. Nearly three years ago we published a paper describing a computational model of the H4–H5 loop  of Na+/K+-ATPase. This model, and especially its improvement connected with usage of vibrational spectroscopy , leads to a root mean square deviation of Ca (RMSD) ~1.6 Å with respect to the NMR average structure  and 1.4 Å with respect to the recently published crystal structure at 2.6 Å resolution . RMSD values of NMR structures  and the crystal structure  are all in the range of 1.4 Å and so, apart from several wrongly modeled loop regions, the computational model describes the structure correctly. Our model allowed computer docking of ATP to the N-domain and thus already at that time to identify the amino acids forming the binding pocket. Experiments based on the structural model followed and we were able to describe the binding interaction in more detail [5-7]. E446, F475, K480, Q482, K501, G502 were identified as amino acids forming the binding pocket correctly. Moreover, the aromatic stacking interaction between ATP and F475 together with one hydrogen bond of the NH2 of the adenosine moiety was described as most important for binding . However, F548 was described incorrectly to be close to the binding site and one hydrogen bond was incorrectly modeled with E446 and not with Q482. Molecular modeling proposed the closest distance 3.8 Å between Q482 and ATP and thus the mechanism of participation of this residue in ATP recognition is rather indirect. Nevertheless, on basis of our experiments we could correctly state that F548 does not influence binding of ATP and that the replacement of Q482 with leucine results in a strong inhibition of both ATP binding . The structurally important hydrogen bond between R423 and E472 was proposed at first on basis of our model . Finally, our model had to be corrected after the publication of the NMR-structure only to a small extent and thus for several years it was the most correct structure for designing structural experiments. In spite of the fact that protein modeling is often underestimated, our work shows that molecular modeling in combination with vibrational spectroscopy and other low resolution techniques is a powerful tool for fast and relatively correct determination of protein structures in general.
This research was supported by the Grant Agency of the Czech Republic (Grants No. 309/02/1479, 206/03/D082), and by the Ministry of Education of the Czech Republic (Grants no. MSM113100001, LN00A141).
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