Crystallization and X-ray structure of a highly aggregation-prone monobody engineered for high-affinity...

Area of Science:

• Protein engineering
• Structural biology
• Biochemistry

Background:

• Monobodies are engineered protein scaffolds offering an alternative to antibodies.
• Engineering variable loops in monobodies can lead to solubility issues and aggregation.
• Studying the structure of aggregation-prone engineered proteins is challenging.

Purpose of the Study:

• To determine the crystal structure of a monobody (Mb-P') engineered to bind HPPU.
• To investigate a strategy for overcoming solubility and aggregation issues in engineered monobodies.
• To enable structural studies of aggregation-prone protein scaffolds.

Main Methods:

• Crystallization and X-ray diffraction at 2.57 Å resolution.
• Engineering of a monobody (Mb-P') for high-affinity binding to HPPU.
• N-terminal fusion of the monobody with maltose-binding protein (MBP) via an optimized linker.

Main Results:

• The crystal structure of Mb-P' fused to MBP was determined.
• The MBP moiety interacted with and shielded aggregation-prone regions of the monobody.
• The fusion strategy successfully produced monodisperse, diffraction-quality crystals from an aggregation-prone protein.

Conclusions:

• N-terminal MBP fusion is an effective strategy to enhance the solubility and enable structural studies of engineered, aggregation-prone monobodies.
• The MBP fusion acts by sterically masking surfaces prone to aggregation.
• This approach facilitates the structural characterization of engineered protein scaffolds.