Extended Data Fig. 2: Engineering the CAGE toolbox via charge-swap mutations of the NrdJ-1 cages.
From: Distinct phases of cellular signaling revealed by time-resolved protein synthesis
a, Schematic showing engineering strategy used to tune the responsiveness of the CAGE via charge-swap mutations. The interaction between the appended caging unit and the split intein can be strengthened or weakened by manipulating key salt bridges. b, A predicted structure of the NrdJ-1 intein generated via AlphaFold2 modeling, with the amino acids for charge-swap mutations in the cages marked in green circles. c, The amino acid sequences of both NrdJ-1NCage and NrdJ-1CCage constructs (intein fragment + appended cage) with the sites altered through charge-swap mutations indicated as in b marked in green boxes and underlined in red. d, Schematic of MBP–GFP splice product generated by proximity-triggered CPS between complementary MBP-CAGEN and CAGEC-GFP with rapalog treatment in cells. Flanking extein sequences at the splice site were either native NrdJ-1 (NPC/SEI) or AAA/SAA. e, Immunoblots of the MBP–GFP splice product (SP) in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for 4 hours. Relative splice product levels were screened with every single mutant in the cages of the CAGEN or CAGEC indicated in b and c and the combination of #2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC. f,g, Immunoblots of the MBP–GFP splice product (SP) in HEK293T cells co-expressing MBP-CAGEN and CAGEC-GFP, treated with either DMSO or rapalog (100 nM) for indicated time points (f) or treated with DMSO or increasing dose of rapalog for 4 hours (g). Left—CAGE without charge-swap mutations, NPC/SEI extein sequence. Middle—engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) NPC/SEI extein sequence. Right—engineered CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC), AAA/SAA extein sequence. Data in e–g are representative of n = 3 independent experiments. CAGEN refers to ‘NrdJ-1NCage-FKBP’, and CAGEC refers to ‘FRB-NrdJ-1CCage’ in e–g. Importantly, the engineered NrdJ-1 CAGE (#2E/#5A in the cage of CAGEN and #1K in the cage of CAGEC) was found to be optimal and used in all subsequent studies.