Antibody fragments, including Fab and Fv fragments, are known to be effective to stabilise and crystallise membrane proteins. However, it has been difficult to raise monoclonal antibodies to recognise conformational epitopes of native membrane proteins using the conventional mouse hybridoma system. We have recently succeeded to raise antibodies against native membrane proteins using the system combined with improved immunization and screening methods.  In my talk, I will present several successful examples of crystallisation of mammalian membrane proteins, including human A_2A adenosine receptor (A_2AAR), using antibody Fab and Fv fragments.

We have successfully raised a mouse monoclonal antibody against human A_2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket. The structure of A_2AAR in complex with the antibody Fab fragment (Fab2838) reveals that Fab2838 recognizes the intracellular surface of A_2AAR and that its complementarity- determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein carboxy-terminal fragment in the active opsin structure and to CDR-3 of the nanobody in the active β₂-adrenergic receptor structure, but locks A_2AAR in an inactive conformation. These results suggest a new strategy to modulate the activity of G-protein-coupled receptors. In the meeting, I will also present successful applications of antibody crystallisation for mammalian transporters.