8F0E

N-terminal WD40 domain of beta'-COPI subunit with four chains in the asymmetric unit

Summary for 8F0E

• Entry DOI: 10.2210/pdb8f0e/pdb
• Related: 8EVL 8EWX
• Descriptor: Coatomer subunit beta' (2 entities in total)
• Functional Keywords: retrograde trafficking, coatomer, copi, cytosolic protein
• Biological source: Saccharomyces cerevisiae (baker's yeast)
• Total number of polymer chains: 4
• Total formula weight: 137522.92

Authors

Dey, D.,Hasan, S.S. (deposition date: 2022-11-02, release date: 2023-08-30, Last modification date: 2023-10-04)

Primary citation

Dey, D.,Hasan, S.S.
Strategies for rapid production of crystallization quality coatomer WD40 domains.
Protein Expr.Purif., 212:106358-106358, 2023

PubMed Abstract: The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and β'-COPI subunits to bind these membrane protein "clients" for ER retrieval. These "αWD40" and "β'WD40" domains are structural homologs that demonstrate distinct selectivity for client proteins. However, elucidation of the atomic-level principles of coatomer-client interactions has been challenging due to the tendency of αWD40 domain to undergo aggregation during expression and purification. Here we describe a rapid recombinant production strategy from E. coli, which substantially enhances the quality of the purified αWD40 domain. The αWD40 purification and crystallization are completed within one day, which minimizes aggregation losses and yields a 1.9 Å resolution crystal structure. We demonstrate the versatility of this strategy by applying it to purify the β'WD40 domain, which yields crystal structures in the 1.2-1.3 Å resolution range. As an alternate recombinant production system, we develop a cost-effective strategy for αWD40 production in human Expi293 cells. Finally, we suggest a roadmap to simplify these protocols further, which is of significance for the production of WD40 mutants prone to rapid aggregation. The WD40 production strategies presented here are likely to have broad applications because the WD40 domain represents one of the largest families of biomolecular interaction modules in the eukaryotic proteome and is critical for trafficking of host as well as viral proteins such as the SARS-CoV-2 spike protein.

PubMed: 37625737
DOI: 10.1016/j.pep.2023.106358

Experimental method

X-RAY DIFFRACTION (1.31 Å)