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Abstract
Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2'-deoxy-ATP, 3'-deoxy-ATP, and inosine 5'-triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2'-OH, the 3'-OH, and the amino group of adenine reduce the extent of binding-induced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups. Phosphorylation of the ATPase was observed for ITP and 2'-deoxy-ATP, but not for 3'-deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa(2) also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.
Highlights
Phosphorylation of the ATPase was observed for inosine 5Ј-triphosphate (ITP) and 2-deoxy-ATP, but not for 3-deoxy-ATP
Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca2؉-ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2deoxy-ATP, 3-deoxy-ATP, and inosine 5-triphosphate), which were modified at specific functional groups of the substrate
We employ a different approach to probe the role of single functional groups of a ligand in the interaction with a protein: using IR spectroscopy we monitored the protein conformational change induced by binding of substrate analogues, which are modified at specific functional groups of the substrate
Summary
Phosphorylation of the ATPase was observed for ITP and 2-deoxy-ATP, but not for 3-deoxy-ATP. Protein and ligand flexibility are important determinants of the interaction and often lead to ligand binding modes that are not anticipated from structures obtained with other ligands To these “failure(s) of the rigid receptor hypothesis” [1] is added here an impressive example: induced-fit binding of nucleotides to the Ca2ϩ-ATPase. We employ a different approach to probe the role of single functional groups of a ligand in the interaction with a protein: using IR spectroscopy we monitored the protein conformational change induced by binding of substrate analogues, which are modified at specific functional groups of the substrate This identifies those functional groups that are important in the interaction with the protein; structure-interaction relationships are obtained that are similar to structure-activity relationships in drug development that relate the chemical structure of compounds to their pharmacological activity
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