A nanobody toolbox targeting dimeric coiled-coil modules for functionalization of designed protein origami structures

Area of Science:

• Protein engineering
• Structural biology
• Nanobiotechnology

Background:

• Coiled-coil (CC) dimers are fundamental building blocks in protein design due to their predictable assembly.
• Protein origami utilizes CC segments for self-assembly into defined polyhedral structures like tetrahedra and prisms.
• Functionalizing these protein scaffolds is crucial for expanding their applications.

Purpose of the Study:

• To develop and characterize nanobodies for targeted functionalization of de novo designed protein origami.
• To demonstrate the versatility of nanobodies in recognizing CC modules across different protein architectures.
• To explore the potential of nanobodies for creating allosteric control in protein structures.

Main Methods:

• Generation of a nanobody library against CC modules of a designed protein origami tetrahedron.
• Characterization of nanobody binding to CC modules in various contexts (dimers, tetrahedra, prisms, bipyramids).
• Structural analysis using X-ray crystallography and small-angle X-ray scattering (SAXS) to elucidate binding mechanisms.

Main Results:

• Nanobodies successfully recognized CC modules in diverse protein origami structures, enabling targeted functionalization.
• Crystal structures revealed nanobody binding within the tetrahedral cavity, interacting with CC dimers.
• Allosteric nanobodies exhibiting positive cooperativity were identified, demonstrating coupled binding events.

Conclusions:

• A toolbox of nanobodies specific for CC modules offers precise targeting of designed protein structures.
• This approach significantly enhances the functionalization capabilities of protein origami scaffolds.
• The developed nanobodies open new avenues for creating advanced CC-based bionanomaterials and functional protein architectures.