Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones.

Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~ 10 nM) for the Fc region of the IgG, but a much lower affinity (KD ~ low εM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 10,000-fold change in affinity from pH 7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.