Three-dimensional structure of the large cytoplasmic H-4-H-5 loop of Na+/K+-ATPase deduced by restraint-based comparative modeling shows only one ATPbinding site

Homology modeling of the complete structure of the large cytoplasmic loop b etween the fourth and fifth transmembrane segments (H-4-H-5 loop) of the al pha subunit of Na+/K+-ATPase is reported. The deduced amino acid sequence s hows high sequence identity and homology to the Ca2+-ATPase (32.8% identity and 53.3% similarity in our alignment), whose tertiary structure has been solved recently at 2.6-Angstrom resolution by X-ray crystallography. This h igh homology allowed the construction of a model structure using the MODELL ER program. Refinement was achieved through interactive visual and algorith mic analysis and minimization with the TRIPOS force field included in the S YBYL/MAXIMIN2 module. The docking of ATP as a substrate into the active sit e of the model was explored with the AUTODOCK program followed by molecular mechanics optimization of the most interesting complexes. Thus, the dockin g of ATP into the resulting model of the H-4-H-5 loop gave evidence for the existence of one ATP binding site only. We were able to specify Cys(549), Phe(548), Glu(505), Lys(501), Gln(482), Lys(480), Ser(477), Phe(475) and Gl u(446) as parts of the ATP binding site with Lys(501) located in the depth of the positively charged binding pocket.