X-ray Crystal Structure Analysis of VHH-Protein Antigen Complexes

VHHs are antigen-binding domains cloned from heavy-chain antibodies found in camelids. These proteins have generated considerable interest in a variety of applications as research reagents, crystallization chaperones, and therapeutics. The evolutionary adaptations of VHHs have resulted in biophysical properties and antigen-binding modalities which are distinct from those of conventional antibodies. A detailed molecular analysis of VHH interactions with their cognate protein antigens is valuable for understanding structure-function relationships and for protein engineering. The majority of VHHs bind to folded proteins and thus recognize discontinuous three-dimensional epitopes. While multiple approaches exist for dissecting the interaction between a protein antigen and a VHH, X-ray crystallography remains the highest resolution method available. Here, we provide an updated procedure for determining and analyzing the X-ray structure of a VHH in complex with a protein antigen. We describe the recombinant expression and purification of VHHs and protein antigens, purification and analysis of protein complexes, crystallization, and optimization, X-ray structure determination by molecular replacement, and analysis of the complex.