Specific Mutations Reverse Regulatory Effects of Adenosine Phosphates and Increase Their Binding Stoichiometry in CBS...

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Regulatory cystathionine β-synthase (CBS) domains are prevalent in proteins, but their regulatory mechanisms remain unclear due to structural complexities.
• Microbial inorganic pyrophosphatases (CBS-PPases) are tetrameric enzymes regulated by adenosine phosphates and alarmones, exhibiting complex allosteric inhibition and activation.
• Each CBS-PPase subunit possesses two CBS domains, yet binds only one mono-adenosine derivative molecule cooperatively, suggesting intricate regulatory site interactions.

Purpose of the Study:

• To elucidate the key protein elements governing the allosteric regulation of Desulfitobacterium hafniense CBS-PPase by mono-adenosine phosphates.
• To investigate the molecular basis for the "half-of-the-sites" ligand binding stoichiometry observed in CBS-PPase.

Main Methods:

• Site-directed mutagenesis was employed to substitute seven key residues within the CBS1 domain of D. hafniense CBS-PPase.
• Enzyme activity assays and isothermal titration calorimetry were utilized to characterize the interactions of 11 CBS-PPase variants with regulatory ligands (ATP, ADP, AMP).

Main Results:

• Specific mutations, such as Lys100 replacement, reversed ADP's effect from inhibition to activation.
• Mutations at Lys95 and Gly118 resulted in concentration-dependent shifts in ADP's regulatory effect (activator at low, inhibitor at high concentrations).
• Mutations significantly increased mono-adenosine phosphate binding stoichiometry, effectively doubling it in some variants.

Conclusions:

• Identified critical amino acid residues (Lys100, Lys95, Gly118) that dictate the allosteric response to ADP in CBS-PPase.
• Provided evidence supporting a "two non-interacting pairs of interacting regulatory sites" model for CBS-PPase regulation, explaining the observed ligand binding stoichiometry.