Structural elements of PC2 required for interaction with its helper protein 7B2

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Eukaryotic subtilisin proteases share structural similarities but differ in activation requirements.
• Prohormone convertase 2 (PC2) is unique in its dependence on the 7B2 protein for activation.
• Understanding PC2-7B2 interactions is key to elucidating PC2 maturation and function.

Purpose of the Study:

• To investigate the specific roles of individual domains within prohormone convertase 2 (PC2) in its interaction with the 7B2 protein.
• To identify the molecular determinants responsible for 7B2 binding and subsequent PC2 activation.

Main Methods:

• Utilized sequential deletions, site-directed mutagenesis, and domain swapping between PC2 and PC1.
• Expressed chimeric and mutant PC2 constructs in AtT-20 cells.
• Analyzed 7B2 binding, protein maturation, and enzymatic activity of modified PC2 variants.

Main Results:

• The PC2 proregion is necessary, but not sufficient, for 7B2 binding.
• The PC2 P domain is essential for structural stability and not interchangeable with PC1's P domain.
• The carboxyl-terminal domain does not participate in 7B2 binding.
• A Tyr-194 to Asp mutation in the catalytic domain abolished 7B2 binding and PC2 activation.

Conclusions:

• The PC2 proregion and a specific residue (Tyr-194) in the catalytic domain are critical for 7B2 interaction.
• A surface-exposed aromatic-rich loop containing Tyr-194 in the catalytic domain likely serves as the primary 7B2 recognition site.
• These findings provide insights into the molecular basis of PC2 activation by 7B2.