Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin...

Area of Science:

• Biochemistry
• Structural Biology
• Immunology

Background:

• Mannose-binding lectin-associated serine proteases (MASP-1 and MASP-2) are crucial for the complement lectin pathway.
• Previous MASP inhibitors were derived from the sunflower trypsin inhibitor (SFTI) peptide, creating SFTI-based MASP inhibitors (SFMIs).

Purpose of the Study:

• To elucidate the structural basis of MASP-1 inhibition by SFMI1.
• To investigate structural requirements for SFMI2 inhibition of MASP-2.
• To develop stable, potent MASP-2 inhibitors for potential therapeutic applications.

Main Methods:

• Crystal structure analysis of the MASP-1/SFMI1 complex.
• Directed evolution of SFTI to enhance plasticity for MASP inhibition.
• Systematic modification of SFMI2, including N- and C-terminal capping and disulfide bridge replacement with thioether linkers.

Main Results:

• The crystal structure revealed that a P2 Thr residue in wild-type SFTI hinders binding to MASPs due to gatekeeper loops.
• Directed evolution by replacing P2 Thr with Ser created structurally plastic SFMIs essential for MASP inhibition.
• A reduction-resistant SFMI2 variant with a l-2,3-diaminopropionic acid (Dap) linker exhibited near-native potency against MASP-2.

Conclusions:

• Structural plasticity, achieved through directed evolution, is critical for effective SFMI inhibition of MASPs.
• Modified SFMI2 variants demonstrate high potency and stability, suggesting potential as therapeutic agents.
• Selective MASP-2 inhibitors could be valuable drug candidates for treating thrombosis associated with COVID-19.