Mapping interactions between the Ca2+-ATPase and its substrate ATP with infrared spectroscopy

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is crucial for muscle contraction.
• Understanding nucleotide binding and phosphorylation is key to SERCA function.
• Previous studies suggested uniform ATPase conformations, but nucleotide interactions remain complex.

Purpose of the Study:

• To investigate the impact of specific nucleotide modifications on Ca(2+)-ATPase conformational changes.
• To determine the relationship between nucleotide binding-induced conformational changes and phosphorylation.
• To elucidate the structural basis of nucleotide-protein interactions in Ca(2+)-ATPase.

Main Methods:

• Utilized infrared spectroscopy to monitor conformational changes in Ca(2+)-ATPase.
• Employed ATP and modified ATP analogues (2'-deoxy-ATP, 3'-deoxy-ATP, ITP) as substrates.
• Analyzed binding-induced conformational changes and phosphorylation efficiency.

Main Results:

• Modifications at the 2'-OH, 3'-OH, and adenine amino group reduced conformational changes.
• The 3'-OH and adenine modifications had the most significant impact on conformational change.
• Phosphorylation occurred with ITP and 2'-deoxy-ATP, but not 3'-deoxy-ATP.
• No direct correlation was found between conformational change extent and phosphorylation rate.
• The conformation of the phosphorylated intermediate (E1PCa(2)) was nucleotide-dependent.

Conclusions:

• Specific nucleotide functional groups are critical for Ca(2+)-ATPase conformational changes.
• Full ATP-induced conformational changes are not essential for phosphorylation.
• ATPase conformational states are more variable than previously thought, influenced by nucleotide binding.