Strategies for rapid production of crystallization quality coatomer WD40 domains

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.

Keywords: Coatomer WD40 domains; E. coli and human Expi293 cells; Expression and purification; Fluorescence size exclusion chromatography; X-ray crystallography.

Figure 1:

E. coli BL21 (DE3) pLysS is a suitable host for the expression of αWD40 domain. (A) Bidirectional trafficking of type I membrane proteins (yellow) and their retrieval by COPI (orange). (B) Schematic diagram of the coatomer complex interacting with client protein dibasic motif (cyan circles). (C) Structural superposition of αWD40 (pink) and β’WD40 (green) domains. N- and C-termini, blue, red spheres. (D) Growth of E. coli BL21 (DE3) pLysS (red) and E. coli BL21 (DE3) (black) cells upon transformation with pSHSαWD40Sp327 plasmid. Standard deviation of triplicates is shown. (E) SEC analysis of strep-HisX6-SUMO-αWD40. (F) SDS-PAGE analysis of purification fractions. Key: 1, molecular weight ladder (kDa); 2, strep-HisX6-SUMO-αWD40 (pull-down); 3, strep-HisX6-SUMO-αWD40 (after Ulp1 digestion); 4, tag-less αWD40 (after SEC, E. coli); 5, αWD40-strep (after SEC, Expi293 cells); 6, tag-less β’WD40 (after SEC, E. coli; included for reference). (G-I) SEC chromatograms showing optimization of strep-HisX6-SUMO-αWD40 digestion by Ulp1. SEC column: HiLoad 16/600 Superdex 75. Equal starting amounts of strep-HisX6-SUMO-αWD40 domain were used in panels (G-I). Amount of Ulp1 protease used per mg of fusion protein: 20 units (panel G), 15 units (panel H), 20 units (panel I).

Figure 2:

PEI is an effective transfection reagent for αWD40 expression in Expi293 cells. (A) SEC analysis of αWD40-strep domain from Expi293 cells transfected with Expifectamine. (B) SEC analysis of αWD40-strep domain from Expi293 cells transfected with PEI. SEC column: HiLoad 16/600 Superdex 75. Equal 200 ml Expi293 cell culture volumes were used in both experiments.

Figure 3:

The β’WD40 domain is more stable when expressed in E. coli BL21 (DE3) pLysS cells than the ɑWD40 domain. (A) SDS-PAGE analysis of purification fractions. Lanes: 1, molecular weight ladder (kDa); 2, strep-HisX6-SUMO-β’WD40 (pull-down); 2, β’WD40 (after Ulp1 digestion); 3, tag-less β’WD40 (after SEC); 4, tag-less αWD40 (after SEC, E. coli); 5, tag-less ɑWD40 for reference. (B) SEC analysis of strep-HisX6-SUMO-β’WD40. (C, D) SEC chromatograms showing release of tag-less β’WD40 from strep-HisX6-SUMO-β’WD40 upon digestion by Ulp1. SEC column: HiLoad 16/600 Superdex 75.

Figure 4:

On-bead cleavage accelerates the purification of the coatomer WD40 domains. (A) SDS-PAGE analysis of purification fractions. Key: 1, molecular weight ladder (kDa); 2, strep-HisX6-SUMO-αWD40 (after Ulp1 digestion); 3, tag-less αWD40 (after SEC); 4, strep-HisX6-SUMO-β’WD40 (after Ulp1 digestion); 5, tag-less β’WD40 (after SEC). (B) SEC analysis of strep-HisX6-SUMO-αWD40 (after Ulp1 digestion). (C) SEC analysis of strep-HisX6-SUMO-β’WD40 (after Ulp1 digestion). SEC column: HiLoad 16/600 Superdex 75.

Figure 5:

Freezing and thawing partially destabilize coatomer WD40 domains. (A) SEC chromatogram of the tag-less αWD40 domain from conventional FPLC. (B) FSEC analysis of the fast thawed tag-less αWD40 domain showing loss of fluorescence intensity in replicates (red, blue, and green) relative to the fresh tag-less αWD40 domain (black). (C) FSEC analysis of the slow thawed tag-less αWD40 domain showing minimal loss of fluorescence intensity in replicates (red, blue, and green) relative to the fresh tag-less αWD40 domain (black). (D) SEC chromatogram of the tag-less β’WD40 domain from conventional FPLC. (E) FSEC analysis of the fast thawed tag-less β’WD40 domain showing loss of fluorescence intensity in replicates (red, blue, and green) relative to the fresh tag-less αWD40 domain (black). (F) FSEC analysis of the slow thawed tag-less β’WD40 domain showing loss of fluorescence intensity in replicates (red, blue, and green) relative to the fresh tag-less αWD40 domain (black). FSEC flow-rate=0.5ml/min. Chromatography column: Superdex 200 Increase 10/300 GL.

Figure 6:

The rapid purification protocol yields high-resolution αWD40 and β’WD40 crystal structures. (A) Crystals of the tag-less αWD40 domain. (B) Crystals of the tag-less β’WD40 domain. (C) Representative electron density in the 1.9Å resolution crystal structure of the tag-less αWD40 domain. Residues: serine-260-cysteine-cysteine-leucine-phenylalanine-264; blue mesh: 2Fo-Fc map, σ=1.0. (D) Representative electron density in the 1.2Å resolution crystal structure of the tag-less β’WD40 domain. Residues: asparagine-188tyrosine-valine-aspartate-tyrosine-192; blue mesh: 2Fo-Fc map, σ=1.0.