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. Author manuscript; available in PMC: 2022 Sep 16.
Published in final edited form as: Cell Chem Biol. 2021 Mar 4;28(9):1283–1297.e8. doi: 10.1016/j.chembiol.2021.02.005

Figure 1. Yeast cell surface display to select for HECT domain mutants of Rsp5 that can pair with xUba1 to transfer xUB.

Figure 1.

(A) Crystal structure of Rsp5 HECT domain showing the loop consisting of residues V606, L607, D608 and T610 that would interact with the N-terminal helix of Ubc1. N632 of the HECT domain may also contribute to E2 interactions. We thus randomized these residues to construct the HECT domain library. (B) Yeast cell surface display for the selection of variants of Rsp5 HECT domain that can pair with xUbc1 for the transfer of xUB. The HECT domain library of Rsp5 was displayed on yeast cell surface as fusions with the yeast protein Aga2p (Chao et al., 2006). N-terminal tagged ybbR-xUB was labeled with biotin-CoA by ybbR tag modification catalyzed by Sfp phosphopantetheinyl transferase (Yin et al., 2006). xUba1 would load biotin-labelled ybbR-xUB (biotin-xUB) onto xUbc1 and the generated xUB~xUbc1 thioester conjugate would react with the Rsp5 HECT domain library displayed on the yeast cell surface. Mutants of HECT domain compatible with xUbc1 in xUB transfer would be covalently conjugated to biotin-xUB and respective yeast cells would be labeled with biotin. After the reaction was done, the cells were washed to remove the free biotin-xUB, xUba1 and xUbc1 enzymes, and were labeled with streptavidin conjugated with the fluorescence protein phycoerythrin (PE). In parallel, a mouse anti-HA antibody and an anti-mouse IgG antibody conjugated with Alexa Flour 647 was added to label the cells expressing the HECT domain with an N-terminal HA tag. Yeast cell library was sorted with a dual color fluorescence gate to select for cells with both abundant expression of HECT and biotin-xUB attachment to the HECT domain to enforce the selection of xHECT domains capable of pairing with xUbc1 in the transfer of xUB. (C) Round of fluorescence-activated cell sorting (FACS) with increasing stringency in reaction conditions and narrower gates of cell collection to enrich clones displaying the HECT mutants conjugated with biotin-xUB. Cells were sorted along the axes of fluorescence from streptavidin-PE and anti-mouse IgG-Alexa Fluor 647 to select for catalytically active xHECT mutants. Percentages in each round of sorting designate the fraction of cells in the Q2 region that are positive in both fluorescence channels. (D) After six rounds of selection, the alignment of 30 clones sequenced shows the convergency of the library with YW01 appearing 18 times and YW04 appearing 4 times.