A designed ankyrin repeat protein evolved to picomolar affinity to Her2.

Area of Science:

• Protein Engineering
• Biochemistry
• Structural Biology

Background:

• Designed ankyrin repeat proteins (DARPins) are versatile binding molecules.
• Previous DARPins showed nanomolar affinity for human epidermal growth factor receptor 2 (Her2).

Purpose of the Study:

• To improve the affinity of DARPins against Her2 through affinity maturation.
• To investigate the structural basis for enhanced Her2 binding.

Main Methods:

• Affinity maturation using error-prone PCR and ribosome display.
• Crystal structure determination of the high-affinity DARPin at 1.7 Å.
• Analysis of Her2 detection sensitivity in human carcinoma extracts.

Main Results:

• A DARPin variant with four framework mutations achieved 90 pM affinity, a 3000-fold increase.
• The highest affinity DARPin exhibited a 500-fold increase in on-rate.
• Structural analysis revealed a His52Tyr mutation altering inter-repeat H-bonding and subdomain disposition, enhancing the on-rate.

Conclusions:

• Affinity maturation effectively increased DARPin binding to Her2.
• The His52Tyr mutation stabilizes a conformationally selected state, boosting binding kinetics.
• Framework residue randomization offers potential for broader DARPin applications and affinities.