4POY: Engineered Dual Specific Vhh Antibody

To explore dual-specificity in a small protein interface, we previously generated a 'metal switch' anti-RNase A VHH antibody using a combinatorial histidine library approach. While most metal-binding sites in proteins are found within rigid secondary structure, the engineered VHH antibody (VHHmetal), which contained three new histidine residues, possessed metal-binding residues within the flexible hypervariable loops. Here, crystal structure analysis of the free and bound states of VHHmetal reveals the structural determinants leading to dual-function. Most notably, CDR1 is observed in two distinct conformations when adopting the metal or RNase A bound states. Furthermore, mutagenesis studies revealed that one of the engineered residues, not located in the metal-binding pocket, contributed indirectly to metal recognition, likely through influencing CDR1 conformation. Despite these changes, VHHmetal possesses a relatively minor energetic penalty toward binding the original antigen, RNase A ( approximately 1 kcal/mol), where the engineered gain-of-function metal-binding residues are observed to possess a mix of favorable and unfavorable contributions towards RNase A recognition. Ultimately, the conformationally distinct metal-switch interface architecture reflects the robust, library-based strategy used to produce VHHmetal. These results also suggest that even small protein interfaces, such as VHH, may be structurally and energetically forgiving in adopting novel function, while maintaining original function.
PDB ID: 4POYDownload
MMDB ID: 123433
PDB Deposition Date: 2014/2/26
Updated in MMDB: 2014/10
Experimental Method:
x-ray diffraction
Resolution: 1.5  Å
Source Organism:
Similar Structures:
Biological Unit for 4POY: monomeric; determined by author and by software (PISA)
Molecular Components in 4POY
Label Count Molecule
Protein (1 molecule)
Molecule annotation
* Click molecule labels to explore molecular sequence information.

Citing MMDB