Structural Basis of High-Precision Protein Ligation and Its Application

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Engineering

Background:

• Enzyme-catalyzed protein modifications offer superior precision and compatibility compared to chemical methods.
• Existing ligases like sortase A and OaAEP1 have limitations in target specificity and application in complex biological environments.
• Connectase, a repurposed protease, showed promise for protein ligation but lacked processivity and structural data for optimization.

Purpose of the Study:

• To elucidate the structural basis of MmConnectase (MmCET) activity through X-ray crystallography.
• To engineer Connectase for enhanced precision and processivity in protein ligation.
• To enable high-precision protein conjugation in challenging biological milieu.

Main Methods:

• X-ray crystallography of MmConnectase in apo and substrate-bound states.
• Comparative structural analysis with MjCET to identify key functional differences.
• Site-directed mutagenesis targeting N-terminal substrate recognition motifs.
• Single-molecule protein unfolding experiments to assess ligation efficiency and processivity.

Main Results:

• Determined the X-ray crystal structures of MmConnectase, revealing the structural basis for its ligation activity.
• Identified key structural features distinguishing MmConnectase from its inactive paralogue, MjCET.
• Proposed and validated modifications to suppress protease activity and enhance ligation precision.
• Demonstrated stepwise tandem ligations using optimized Connectase and OaAEP1(C247A) for polymer formation.

Conclusions:

• Structural insights into MmConnectase facilitate its engineering for advanced protein ligation.
• Modified Connectase enables high-precision protein conjugation in complex biological environments like cell cultures.
• Optimized Connectase demonstrates enhanced processivity for creating defined protein polymers.