Llama Single Domain Antibody Contributes to Crystal Structure and Nobel Prize
In 2012, Brian K. Kolbika and Robert J. Lefkowitz were awarded the Nobel Prize in Chemistry for their investigation of G-Protein Coupled Receptors. Kolbika and Lefkowitz did extensive studies that allow us an understanding of the molecular mechanism of how G-Protein Coupled Receptors (GPCRs) work.
Extracellular cues are commonly converted into specific intracellular responses through the activity of G-protein coupled receptors. This mediation is paramount to regulating the majority of physiological processes. As a result, the ligands responsible for GPCR signaling have the potential to be used as models for a novel class of therapeutics. The binding of agonists to the orthosteric pocket of a GPCR stabilizes the specific receptor conformation which results in binding of three separate protein groups: first heterotrimeric GTP-binding proteins (G-proteins), second G-protein coupled receptor kinases (GRK’s) and finally β-arrestins. Subsequently, biological activity of the GPCRs is regulated by the stabilization of specific conformations that either activate or inhibit.
Seven-transmembrane receptors (7MTRs), the largest class of receptors found in the human genome, are frequently the target for medicinal therapeutics. Lefkowitz identified β-arrestin as true adaptor proteins that mediate signaling for several effector pathways. The biochemical and functional consequences of signals transduced by β-arrestins are distinct from those transduced by G proteins. Ligands and receptors have been isolated that preferentially signal through pathways mediated by either G-proteins or β-arrestins. The ligands responsible for 7TMR signaling could be used as models for a novel class of therapeutics.
Ligand binding to orthosteric or allosteric sites on GPCRs induces biological activity through the stabilization of specific receptor conformations. These ligands are typically small molecule agonists and antagonists. Lefkowitz identified monomeric single domain antibodies (single domain antibodies) from the Camelid family that were capable of allosterically binding to and stabilizing an active conformation of the β2 adrenergic receptor (β2AR). The lab characterized a family of sequence related single domain antibodies with preferential binding to active (agonist occupied) or inactive (antagonist occupied) receptors. Additional studies revealed that the single domain antibodies bound to epitopes found on the intracellular receptor surface. When the single domain antibodies were expressed intracellularly, conformational specificity was preserved, and the single domain antibodies selectively bound agonist/antagonist-occupied receptors. The “intrabodies” (intracellularly expressed single domain antibodies) acted to inhibit G-protein activation (cyclic AMP accumulation), GRK-mediated receptor phosphorylation, β-arrestin recruitment and receptor internalization. Lefkowitz postulated that the measured functional effects were due to the stabilization of specific receptor conformations.
The two teams of investigators utilized five strategies to produce a stable ternary complex ideally suited for crystallization. One of the successful strategies relied upon the selection of a camelid single domain antibody that allowed for stabilization of the Gβ-subunit in a well-defined conformation.