Structural insight into an anti‐BRIL Fab as a G‐protein‐coupled receptor crystallization chaperone

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 79; no. 5; pp. 435 - 441
• Main Authors: Miyagi, Hikaru, Suzuki, Michihiko, Yasunaga, Mai, Asada, Hidetsugu, Iwata, So, Saito, Jun-ichi
• Format: Journal Article
• Language: English
• Published: 5 Abbey Square, Chester, Cheshire CH1 2HU, England International Union of Crystallography 01.05.2023; Wiley Subscription Services, Inc
• Subjects: Antibodies; anti‐BRIL Fabs; apocytochrome b562; Binding; crystal packing; Crystal structure; Crystallization; crystallization chaperones; Drug delivery; Drug development; E coli; Epitopes; Escherichia coli; Fab; Fusion protein; G protein-coupled receptors; GPCRs; Helices; Immunoglobulin Fab Fragments - chemistry; Membrane proteins; Protein structure; Protein Structure, Secondary; Proteins; Receptors; Receptors, G-Protein-Coupled - chemistry; Stacking; Therapeutic targets; crystal packing; crystal structure; crystallization chaperones; apocytochrome b562; anti-BRIL Fabs; GPCRs
• ISSN: 2059-7983; 0907-4449; 2059-7983; 1399-0047
• DOI: 10.1107/S205979832300311X

Structure determination of G‐protein‐coupled receptors (GPCRs) is key for the successful development of efficient drugs targeting GPCRs. BRIL is a thermostabilized apocytochrome b562 (with M7W/H102I/R106L mutations) from Escherichia coli and is often used as a GPCR fusion protein for expression and crystallization. SRP2070Fab, an anti‐BRIL antibody Fab fragment, has been reported to facilitate and enhance the crystallization of BRIL‐fused GPCRs as a crystallization chaperone. This study was conducted to characterize the high‐resolution crystal structure of the BRIL–SRP2070Fab complex. The structure of the BRIL–SRP2070Fab complex was determined at 2.1 Å resolution. This high‐resolution structure elucidates the binding interaction between BRIL and SRP2070Fab. When binding to BRIL, SRP2070Fab recognizes conformational epitopes, not linear epitopes, on the surface of BRIL helices III and IV, thereby binding perpendicularly to the helices, which indicates stable binding. Additionally, the packing contacts of the BRIL–SRP2070Fab co‐crystal are largely due to SRP2070Fab rather than BRIL. The accumulation of SRP2070Fab molecules by stacking is remarkable and is consistent with the finding that stacking of SRP2070Fab is predominant in known crystal structures of BRIL‐fused GPCRs complexed with SRP2070Fab. These findings clarified the mechanism of SRP2070Fab as a crystallization chaperone. Moreover, these data will be useful in the structure‐based drug design of membrane‐protein drug targets. The structure of a complex between BRIL and an anti‐BRIL antibody (SRP2070Fab) has been determined at high resolution. This work presents a detailed elucidation of the interaction between BRIL and SRP2070Fab, which may help to improve the function of SRP2070Fab as a crystallization chaperone for membrane proteins.