Affinity- and specificity-enhancing mutations are frequent in multispecific interactions between TIMP2 and MMPs

Area of Science:

• Protein engineering and computational biology
• Molecular interactions and drug design

Background:

• Multispecific proteins are crucial for cellular functions, including signaling and immune response.
• Understanding atomic-level interactions of multispecific proteins is vital for basic science and drug development.
• Tissue Inhibitor of Matrix Metalloproteinases 2 (TIMP2) is a multispecific protein that binds over 26 Matrix Metalloproteinase (MMP) family members.

Purpose of the Study:

• To investigate the hypothesis that TIMP2's multispecificity results in a binding interface not optimized for any single Matrix Metalloproteinase (MMP) target.
• To explore the potential for enhancing TIMP2 binding affinity and specificity through computational and experimental methods.

Main Methods:

• Computational saturation mutagenesis of the TIMP2 binding interface.
• Prediction of binding free energy changes for eight MMP targets.
• Experimental verification of selected TIMP2 point mutants for enhanced binding to MMP14.

Main Results:

• Computational analysis revealed that the TIMP2 binding interface is not optimized for any of the studied Matrix Metalloproteinases (MMPs).
• Numerous affinity-enhancing mutations were identified at various positions within the TIMP2 binding interface.
• Experimental validation confirmed that several TIMP2 point mutations significantly improve binding affinity (over tenfold) and specificity towards MMP14.

Conclusions:

• The TIMP2 protein sequence is suboptimal for binding its target enzymes, indicating substantial room for affinity and specificity enhancement.
• The observed non-optimality and potential for improvement in TIMP2 may be a common characteristic of proteins evolved for multispecific interactions.