Quantitative reactivity profiling predicts functional cysteines in proteomes

Abstract

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

Figure 1. A quantitative approach to globally profile cysteine reactivity in proteomes

a, isoTOP-ABPP involves proteome labeling, click chemistry-based incorporation of isotopically-labeled cleavable tags, and sequential on-bead protease digestions to afford probe-labeled peptides for MS analysis. The IA-probe is shown in the inset. b, Measured isoTOP-ABPP ratios for peptides from MCF7 cells labeled with four pairwise IA-probe concentrations (10:10 μM, 20:10 μM, 50:10 μM, 100:10 μM). The blue box highlights peptides with low isoTOP-ABPP ratios (R < 2.0). Chromatographs for CKB (low ratio) and LCP1 (high ratio) are shown, with elution profiles for heavy- and light-labeled peptides in blue and red, respectively, and green lines depicting peak-boundaries used for quantitation. Isotopic envelopes are shown for light- and heavy-labeled peptides with green lines representing predicted values. Additional chromatographs from isoTOP-ABPP experiments are in Supplementary Table 7.

Figure 2. Hyperreactive cysteines are highly enriched in functional residues

a, Chromatographs from an isoTOP-ABPP experiment using 100:10 μM IA-probe are shown for peptides from GSTO1 (top) and ACAT1 (bottom). The cysteine nucleophiles (asterisk) display low ratios (R_10:1 ≈ 1), whereas other cysteines exhibit high ratios (R_10:1 ≥ 4). b, Pie charts illustrating the percentage of functionally annotated cysteines for three isoTOP-ABPP ratio ranges, including an average derived from all cysteines in the UniProt database. c, Correlation of isoTOP-ABPP ratios with functional annotations from the UniProt database where active-site nucleophiles or redox-active disulfides are shown in red, and all other cysteines in black. A moving-average (window of 50) of functional residues is shown as a dashed-blue line, demonstrating a profound enrichment within R_10:1 < 2.0. Data are from experiments in three human cancer cell lines (MCF7, MDA-MB-231 and Jurkat).

Figure 3. Functional characterization of the hyperreactive cysteines in PRMT1

a, Crystal structure of rat PRMT1 (green, PDB: 1ORI) showing the hyperreactive cysteine C101 in contact with an S-adenosylhomocysteine (SAH) cofactor (cyan). b, Wild-type and C101A mutant of human PRMT1 were labeled with the IA-probe, followed by click-chemistry to incorporate a fluorescent rhodamine tag. In-gel fluorescence demonstrates robust labeling of the wild-type (WT) but not C101A mutant PRMT1 and IA-labeling of WT PRMT1 is inhibited by HNE. c, Catalytic activity of purified WT, but not C101A PRMT1 is inhibited by HNE as measured by monitoring transfer of ³H-methyl from ³H-S-adenosylmethionine (SAM) to a histone 4 substrate.

Figure 4. Functional characterization of YHR122W/FAM96B

a, Expression of wild-type (WT) and C161A-YHR122W in a yeast strain with a doxycycline (dox)-repressable YHR122W gene demonstrated a dominant-negative phenotype upon induction of C161A-YHR122W expression (−dox/+gal, middle panel) and rescue of viability by expression of WT, but not C161A-YHR122W (+dox/+gal, right panel). b, The FeS cluster assembly pathway contains multiple proteins with hyperreactive cysteines (in red). YHR122W/FAM96B (YHR) is a putative member of this network based on protein-protein interaction studies (see http://www.yeastgenome.org/). This panel was adapted from reference . c, Doxycycline treatment of the YHR122W-repressable yeast strain significantly decreased the activity of the cytosolic FeS enzyme Leu1, and this activity is rescued by overexpression of WT-YHR122W. These treatments had no effect on the activity of the non-FeS enzyme alcohol dehydrogenase (ADH). ***, P < 0.001, t-test.

Figure 5. Quantitative reactivity profiling predicts functional cysteines in designed proteins

a, In-gel fluorescence demonstrates robust IA labeling of two active cysteine hydrolases, ECH13 and ECH19 , relative to inactive designs (top panel). Hydrolysis activities of ECH13 and ECH19 measured as the ratio of velocities in the presence versus absence of purified enzymes were 71.64+/−6.94 and 104.15+/−10.78, respectively (see Supplementary Fig. 15a for substrate hydrolysis assay). Other designs showed no measurable hydrolysis activity over background (0.76 +/− 0.058 nmol/s). Asterisks designate Coomassie blue signals for protein designs (lower panel). b, IA-labeling is observed for ECH13 and ECH19, but not their active-site cysteine mutants C45A and C161A, respectively. c, Catalytic cysteines in ECH13 and ECH19 demonstrate low isoTOP-ABPP ratios (red) compared with other designs (blue). Chromatographs are shown for peptides from the nine designs identified in this experiment (bottom panel).