Structural changes of the sarcoplasmic reticulum Ca(II)-ATPase nucleotide binding domain by pH and La(III).
The Ca(2+)-ATPase from sarcoplasmic reticulum couples the hydrolysis of one molecule of ATP to the transport of two Ca2+ ions in skeletal muscle fibers. Here, we study the accessibility of the fluorescein covalently attached to the Lys515 at the nucleotide binding domain of the ATPase to the small collisional quencher iodide at pH 6 and 8, as well as the effect of ligand binding (La3+, La(3+)-nucleotide, and Ca2+). Our results indicate that bound fluorescein is significantly more accessible at pH 6 than at pH 8, suggesting that pH modulates the structure of the nucleotide binding domain of the ATPase. This notion was further substantiated by the finding that La(3+)-nucleotide only interacted with the catalytic center at acidic pH. Notably, the differential accessibility of the nucleotide binding domain at acidic and basic pH cannot be rationalized in terms of the ATPase E1/E2 conformational equilibrium since a shift of the ATPase toward the E1 (plus Ca2+) or E2 (plus EGTA) did not affect the accessibility of fluorescein-labeled ATPase to the quencher. Taken together, these findings show the presence of structural flexibility in the FITC binding site and suggest a structural modulation of the Ca(2+)-ATPase nucleotide binding domain by pH and La3+ binding through long-range link-age mechanisms.
Merino
J M
JM
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain.
Henao
F
F
Gutiérrez-Merino
C
C
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007811:Lanthanum / Q000378:metabolism* / Q000494:pharmacology; D008239:Lysine; D008274:Magnesium / Q000494:pharmacology; D018482:Muscle, Skeletal / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
C028521:lanthanum chloride; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D007811:Lanthanum; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D008239:Lysine