Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

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 bindinginduced 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.Ligand binding to proteins controls vast numbers of cellular processes and has attracted great scientific and economic interest. 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 Ca 2ϩ -ATPase. This finding stems from a systematic mapping of substrate-protein interactions with infrared (IR) 1 spectroscopy. New approaches like this are welcome in the field of ligand-protein recognition, since the most informative techniques, NMR and x-ray crystallography, are laborious and problematic for some systems. Methods like fluorescence and luminescence that require less expenditure also provide less molecular information. We expect that this technology gap will be bridged by IR spectroscopy.IR spectroscopy, one of the methods of vibrational spectroscopy, provides direct information on the molecular level, is cost-effective, and can be universally applied from small soluble proteins to large membrane proteins under near-physiological conditions. Work summarized in recent reviews (2-5) has shown that the vibrational spectrum changes characteristically when a ligand binds to a protein. This provides a direct observation of ligand binding: no marker compound has to be introduced to report the binding process, as with many other methods. Previous work has mostly focused on individual interactions between a ligand and a protein by monitoring the ...

Fe2+-catalyzed Oxidative Cleavages of Ca2+-ATPase Reveal Novel Features of Its Pumping Mechanism

“…It is therefore definitely possible that the cleavage in the N-domain could represent the product of close transient associations of the N-and P-domains mediated by the bound AMP-PNP⅐Fe 2ϩ complex, whereas the crystal structure may represent a more stable form in which the N-and P-domains are less close to each other. Interestingly, recent infrared spectroscopy results (10) suggest that nucleotide binding induces a conformation that is characteristic of the bound nucleotide, pointing at a further line of investigation in which future work might be helpful. In any event, the approach of the ATP molecule bound to the N-domain toward the P- 3 The same is true concerning the second Mg 2ϩ binding site reported by Sørensen et al (6), which is located at a distance of about 5 Å from Glu 439 but which so far has only been detected corresponding to the phosphorylated transition state.…”

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

“…FTIR spectroscopy (22-27) can provide potent dynamic and structural information, which sheds light on interactions occurring between enzyme and substrate during the catalytic process. In our previous work (21,28,29), various nucleotides have been used to probe interactions between nucleotides and the SR Ca 2ϩ -ATPase in the nucleotide binding reaction with FTIR spectroscopy, and characteristic binding modes for each nucleotide have been concluded.Here we investigate enzyme phosphorylation with ATP and ATP analogs inosine 5Ј-triphosphate (ITP) and 2Ј-and 3Ј-dATP to identify important functional groups of ATP for ATPase phosphorylation. The analogs differ from ATP at individual functional groups, which allows us to study the impact of these groups on the conformational change in the phosphorylation reaction (Ca 2 E1ATP 3 Ca 2 E1P).…”

Infrared spectroscopy as a new tool for studying single living cells: Is there a niche?

“…The presence of water can cause some problems in the spectral analysis of biological samples, due to its absorptions at ∼3285 cm −1 , ∼2100 cm −1 and ∼1640 cm −1 that could overlap the bands of other components [37,56,57]. In this perspective, Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) is considered the best approach for studying both hydrated and dried biological samples, such as cells and fluids.…”