Abstract
G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them.
Highlights
G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands
We demonstrate that specific binders, such as antibodies, against soluble fusion partners of GPCRs can facilitate GPCR crystallisation
We discovered a new anti-b562 from Escherichia coli M7W/H102I/R106L (BRIL) antibody (SRP2070Fab), which promoted crystallisation of ICL BRIL-fused GPCRs
Summary
G-protein-coupled receptors (GPCRs)—the largest family of cell-surface membrane proteins—mediate the intracellular signal transduction of many external ligands. The electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them. During small molecule drug development, the three-dimensional structure of the target protein with the candidate compound is very useful for refining the compound to improve its binding capacity This method— structure-based drug design (SBDD)—has been widely used since the 1990s4–6. This approach is useful to expand the soluble regions of the target for crystal p acking[11], antibodies must be screened for each target GPCR to identify those with high binding affinity, which is very difficult and laborious Another common approach to increase the thermal stabilities of GPCRs is to mutate the transmembrane h elices[3,11]. The mutation sites are originally specified based on our extensive knowledge of the active and inactive forms of the adenosine A 2a
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