Extended Data Fig. 5. Functional impact of aminophilic compound-lysine interactions for representative proteins.

a, The location of liganded lysine K117 (orange) in the RIDA crystal structure (gray, PDB ID: 1ONI). Also shown is bound pyruvate (teal) in each of the three active sites at the interfaces of adjacent monomers. b, SAR for aminophilic compound engagement of K117 in RIDA, as determined by competitive isoTOP-ABPP is recapitulated by gel-ABPP of recombinant protein (also see Supplementary Datasets 3 and 4). Top, HEK293T cells recombinantly expressing WT-RIDA and the corresponding K117R mutant as Flag epitope-tagged proteins were treated with the indicated aminophilic compounds (50 µM, 1 h) followed by treatment with probe P2 and analyzed by gel-ABPP (top panel) and western blotting (bottom panel). Bottom, Extracted MS1 chromatograms depicting R values for the indicated aminophilic compound-RIDA-K117 interactions mapped by competitive isoTOP-ABPP (also see Supplementary Dataset 3). c, Top, gel-ABPP data showing concentration-dependent blockade of P2 labeling of recombinantly expressed WT-RIDA by 28h and 26l in HEK293T cell lysates. Bottom, structures of 28h and 26l with extracted MS1 chromatograms depicting R values for their respective engagement of K117 or RIDA determined by competitive isoTOP-ABPP (also see Supplementary Dataset 3). d, Corresponding fitted IC₅₀ curves for blockade of probe 2 labeling of WT-RIDA. Data represent average values ± SD; n = 3 per group. CI, confidence interval. e, Representative isoTOP-ABPP ratio plot showing proteome-wide lysine reactivity profile for 26l (50 μM). Among ~3,000 quantified lysines, only two - K117 of RIDA and K1070 of VCL - were liganded. The dashed line marks the R value of 4 used to define a liganded lysine event (also see Supplementary Dataset 3). f, g, Fitted IC₅₀ curves for the concentration-dependent inhibition of the deaminase activity of recombinantly expressed WT- and K117R and K117I mutants of RIDA in HEK293T cell lysates by 28h (f) and 26l (g). Data represent average values ± SD; n = 3 per group. CI, confidence interval. h, Catalytic activity (upper panel) and gel-ABPP analysis of P2 labeling (lower panel) of WT- and indicated K117 mutants. i, Presumed reversible-covalent and irreversible adducts formed between 26l with K117 and R117.⁶ Data represent average values ± SD; n = 3 per group. P values were 0.00081 and 0.000066. For western blot and gel-ABPP data in b, c, and h, experiments were conducted three times (n = 3 biologically independent experiments) with similar results. Statistical significance was calculated for changes >25% in magnitude in comparison to DMSO-treated samples with unpaired two-tailed Student’s t-tests: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.