Phosphorylation of the Sarcoplasmic Reticulum Ca2+-ATPase from ATP and ATP Analogs Studied by Infrared Spectroscopy

Abstract

Phosphorylation of the sarcoplasmic reticulum Ca 2؉ -ATPase (SERCA1a) was studied with time-resolved Fourier transform infrared spectroscopy. ATP and ATP analogs (ITP, 2 -and 3 -dATP) were used to study the effect of the adenine ring and the ribose hydroxyl groups on ATPase phosphorylation. All modifications of ATP altered conformational changes and phosphorylation kinetics. The differences compared with ATP increased in the following order: 3 -dATP > ITP > 2 -dATP. Enzyme phosphorylation with ITP results in larger absorbance changes in the amide I region, indicating larger conformational changes of the Ca 2؉ -ATPase. The respective absorbance changes obtained with 3 -dATP are significantly different from the others with different band positions and amplitudes in the amide I region, indicating different conformational changes of the protein backbone. ATPase phosphorylation with 3 -dATP is also much (ϳ30 times) slower than with ATP. Our results indicate that modifications to functional groups of ATP (the ribose 2 -and 3 -OH and the amino group in the adenine ring) affect ␥-phosphate transfer to the phosphorylation site of the Ca 2؉ -ATPase by changing the extent of conformational change and the phosphorylation rate. ADP binding to the ADP-sensitive phosphoenzyme (Ca 2 E1P) stabilizes the closed conformation of Ca 2 E1P.In skeletal muscle cells the Ca 2ϩ -ATPase of sarcoplasmic reticulum (SR) 1 pumps Ca 2ϩ actively from the cytoplasm into the SR lumen (1-6). ATP hydrolysis supplies the energy for Ca 2ϩ transport. In the reaction cycle of Ca 2ϩ transfer, the ATPase undergoes conformational changes and forms several intermediates including Ca 2 E1 (the calcium-ATPase complex with high affinity to Ca 2ϩ ), Ca 2 E1ATP (the calcium-ATPATPase complex), Ca 2 E1P (the ADP-sensitive phosphoenzyme), E2P (the ADP-insensitive phosphoenzyme), and E2 (the calcium-free ATPase with low affinity to Ca 2ϩ ).Crystal structures of the SR Ca 2ϩ -ATPase (7-10) show that the enzyme has two regions, the transmembrane region and the cytoplasmic region. The cytoplasmic region consists of three globular domains: nucleotide domain (N domain), phosphorylation domain (P domain), and actuator or anchor domain (A domain), which are connected by hinge regions. The adenosine part of ATP binds to the N domain (11), and the phosphorylated residue Asp-351 is located in the P domain (7). It has been proposed that the structures of Ca 2 E1ATP and of Ca 2 E1P are similar, indicated by similar infrared spectra of ATP binding and Ca 2 E1P formation (12) and by a proteolysis study (13). Recently crystal structures of the complex Ca 2 E1AMPPCP and the Ca 2 E1P analogue Ca 2 E1ADP:AlF 4Ϫ were obtained (9, 10). They are almost identical and show a compact arrangement of domains. The aim of this work is to study the conformational difference between the Ca 2 E1ATP and Ca 2 E1P state with Fourier transform infrared (FTIR) spectroscopy.Infrared spectroscopy (14 -18) has several advantages for elucidating the molecular mechanism of proteins, such as high time res...

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