9T9P

Adenosine receptor A2a (A2AR)-beta-lactamase fusion bound to beta-lactamase inhibitory protein II (BLIPII) and ZM241385

Summary for 9T9P

• Entry DOI: 10.2210/pdb9t9p/pdb
• EMDB information: 55723
• Descriptor: Beta-lactamase inhibitory protein II, Adenosine receptor A2a,Small exopenicillinase,Green fluorescent protein, 4-{2-[(7-amino-2-furan-2-yl[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino]ethyl}phenol (3 entities in total)
• Functional Keywords: g-protein coupled receptor, gpcr, fusion tag, membrane protein
• Biological source: Streptomyces exfoliatus; More
• Total number of polymer chains: 2
• Total formula weight: 124365.30

Authors

Shah, N.R.,Bisson, C.,Hutchin, A.,McFarlane, C.R.,Oosterlaken, M.,Pavic, A.,Zebisch, M. (deposition date: 2025-11-24, release date: 2026-05-27)

Primary citation

Shah, N.R.,Oosterlaken, M.,Bisson, C.,Bertinelli, M.,Churchill-Angus, A.M.,Hutchin, A.,Kopec, J.,Kotov, V.,McFarlane, C.R.,Griss Pascualli, E.,Pavic, A.,Zebisch, M.,Fiez-Vandal, C.,Fabini, E.,Duclos, S.
A novel fusion tool to enable G protein-coupled receptor structure determination.
Acta Crystallogr D Struct Biol, 2026

PubMed Abstract: Structure determination of G protein-coupled receptors (GPCRs) plays an important role in accelerating drug development against this medically important protein family. This study outlines the development of a new fusion tool to enable structure determination of GPCRs in inactive conformations by cryo-EM. Initially, a PDB mining approach was applied to select eight naturally occurring proteins with the intention of fusing them into the intracellular loop 3 (ICL3) of GPCRs to create a suitable fiducial marker for cryo-EM workflows. During the selection process, candidates with known high-affinity protein binders were prioritized to enable a further increase in the protein mass of the fiducial marker. Fusion constructs were generated with adenosine receptor A (AR) and were assessed for expression and aggregation levels. For the two best-performing new fusion constructs, ligand binding was characterized to ensure that the fusion tag did not significantly affect protein behaviour. AR with a β-lactamase fusion in ICL3 and binding partner β-lactamase inhibitory protein II (BLIPII) was then selected to solve an antagonist-bound structure. The overall map was resolved to an average of 3.2 Å resolution with continuous helices connecting the β-lactamase to helices 5 and 6 of AR. Focused refinement of the AR region improved the local resolution and map detail in the orthosteric site, thereby allowing confident modelling of the antagonist ligand, ZM241385, which matches previously described X-ray crystallographic structures. This new fusion provides an alternative option for GPCR structure determination, with several potential benefits compared with existing tools, such as a more favourable position relative to the GPCR to reduce potential clashes.

PubMed: 42138221
DOI: 10.1107/S2059798326003785

Experimental method

ELECTRON MICROSCOPY (3 Å)