Mutations affecting the activity of urokinase-type plasminogen activator

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Single-chain urokinase-type plasminogen activator (scu-PA) is a serine protease involved in fibrinolysis.
• Understanding scu-PA's structure-activity relationship is crucial for developing targeted therapies.

Purpose of the Study:

• To identify specific amino acid residues essential for scu-PA activity through mutagenesis.
• To elucidate the structural basis of scu-PA function and identify key residues in its protease domain.

Main Methods:

• Systematic mutagenesis of the scu-PA cDNA molecule.
• Expression of mutant scu-PA in yeast and assessment of secreted protein activity.
• Analysis of mutations within the serine protease encoding domain (B-chain).
• Utilizing a three-dimensional model of the urokinase protease domain to interpret mutation effects.

Main Results:

• Twelve of thirteen identified scu-PA variants exhibited reduced fibrinolytic activity due to single codon alterations.
• Mutations frequently occurred in highly conserved residues within the serine protease domain.
• Structural modeling indicated that detrimental mutations interfere with substrate binding or introduce steric hindrance at critical internal positions.
• Attempts to rescue mutant activity via second-site reversion yielded only same-site restoration.

Conclusions:

• Specific amino acid residues, particularly conserved ones in the serine protease domain, are critical for scu-PA activity.
• The three-dimensional structure provides insights into how mutations impact scu-PA function, affecting substrate binding and molecular stability.
• The active site and surrounding regions are sensitive to alterations, with limited capacity for compensatory mutations elsewhere.