Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Science

Background:

• Ovalbumin, a serpin superfamily member, lacks the characteristic loop-to-sheet conformational change of inhibitory serpins.
• This conformational change is crucial for proteinase inhibition.

Purpose of the Study:

• To investigate if hinge region mutations can transform ovalbumin into a proteinase inhibitor.
• To understand the structural basis for ovalbumin's noninhibitory nature.

Main Methods:

• Engineered ovalbumin variants with specific mutations, including P14 residue changes.
• Assessed proteinase inhibitory properties and loop insertion rates.
• Utilized porcine pancreatic elastase (PPE) for cleavage at the P1-P1' bond.
• Measured thermal stability and susceptibility to further proteolysis to evaluate loop insertion.

Main Results:

• None of the initial three variants exhibited proteinase inhibitory properties.
• Mutations at P14 (R to S) significantly increased the rate of reactive center loop insertion into beta-sheet A after PPE cleavage.
• Cleaved P14 variants showed increased thermal stability, indicating partial loop insertion.
• Further proteolysis at P8-P7 indicated incomplete loop insertion, suggesting a defect in beta-sheet A accommodation.

Conclusions:

• Ovalbumin's inability to be a proteinase inhibitor is linked to a defect in beta-sheet A's ability to fully accommodate the reactive center loop.
• This structural limitation hinders the extent and/or rate of loop insertion, preventing functional inhibition.