Enhancement of Protein-Protein Interactions by Destabilizing Mutations Revealed by HDX-MS

Area of Science:

• Protein engineering
• Biochemistry
• Structural biology

Background:

• Enhancing protein-protein interactions is crucial for therapeutic efficacy.
• Previous studies suggested destabilizing mutations could improve binding affinity.
• This work validates and expands upon this strategy.

Purpose of the Study:

• To demonstrate the generality of enhancing protein-protein interactions by destabilizing unbound proteins.
• To show this strategy can be applied for therapeutic benefit.
• To provide examples of successful implementation in protein therapeutics.

Main Methods:

• Review of existing literature on destabilizing mutations.
• Analysis of experimental data including differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and hydrogen/deuterium exchange-mass spectrometry (HDX-MS).
• Case studies involving engineered human growth hormone (hGHv) and monoclonal antibodies (mAbs) with specific mutations (YTE, JAWA).

Main Results:

• Destabilizing mutations in unbound proteins enhanced binding affinity to their targets (e.g., hGHbp, FcRn).
• Mutations increased the free energy of the unbound state without significantly altering the bound state free energy.
• Examples include a 400-fold binding improvement in hGHv and a 10-fold increase in mAb-FcRn binding (YTE mutations), leading to extended serum half-life.
• JAWA mutations in mAbs facilitated multimerization for enhanced agonism and effector functions.

Conclusions:

• Destabilizing unbound proteins is a viable strategy to enhance protein-protein interactions for therapeutic purposes.
• This approach offers an alternative to traditional interface optimization for improving binding affinity.
• The findings support the rational design of protein therapeutics with improved pharmacokinetic and pharmacodynamic properties.