Engineering gain-of-function mutants of a WW domain by dynamics and structural analysis

Area of Science:

• Protein Engineering
• Computational Biology
• Biophysics

Background:

• Protein sequences designed through evolution ensure foldability and function.
• Classical studies show that sequence alone is insufficient for foldability, requiring consideration of local contacts during folding.
• Previous work demonstrated restoring foldability and function by reducing frustration in a model WW domain protein's folding energy landscape.

Purpose of the Study:

• To computationally design single-point mutations that enable a model protein (N21) to bind a Group I peptide ligand.
• To investigate the roles of specific residues and dynamic profiles in peptide binding affinity.
• To validate the use of dynamic profiles in guiding protein engineering for enhanced binding affinity.

Main Methods:

• Structure and dynamic-based computational design to identify mutations.
• Comparison of docked ligand complex structures with native protein structures.
• Analysis of dynamic profiles to identify allosterically coupled residues.
• Site-directed mutagenesis to swap residues and assess binding affinity.

Main Results:

• Single-point mutations were identified that enable N21 to bind Group I peptides.
• Residues at positions 9 and 19 were found to be crucial for peptide binding.
• Position 10 was identified as allosterically coupled to the binding site, with distinct dynamics between N21 and the engineered CC16-N21.
• Swapping specific residues in N21 with those from CC16-N21 restored native-like binding affinity.

Conclusions:

• Computational design, guided by dynamic profiles, can effectively engineer protein binding affinity.
• Specific residue substitutions and allosteric dynamics are critical for modulating protein-ligand interactions.
• Dynamic profiles serve as valuable guiding principles for the targeted modification of small protein binding affinities.