Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue

Abstract

Arylfluorosulfates have appeared only rarely in the literature and have not been explored as probes for covalent conjugation to proteins, possibly because they were assumed to possess high reactivity, as with other sulfur(VI) halides. However, we find that arylfluorosulfates become reactive only under certain circumstances, e.g., when fluoride displacement by a nucleophile is facilitated. Herein, we explore the reactivity of structurally simple arylfluorosulfates toward the proteome of human cells. We demonstrate that the protein reactivity of arylfluorosulfates is lower than that of the corresponding aryl sulfonyl fluorides, which are better characterized with regard to proteome reactivity. We discovered that simple hydrophobic arylfluorosulfates selectively react with a few members of the intracellular lipid binding protein (iLBP) family. A central function of iLBPs is to deliver small-molecule ligands to nuclear hormone receptors. Arylfluorosulfate probe 1 reacts with a conserved tyrosine residue in the ligand-binding site of a subset of iLBPs. Arylfluorosulfate probes 3 and 4, featuring a biphenyl core, very selectively and efficiently modify cellular retinoic acid binding protein 2 (CRABP2), both in vitro and in living cells. The X-ray crystal structure of the CRABP2-4 conjugate, when considered together with binding site mutagenesis experiments, provides insight into how CRABP2 might activate arylfluorosulfates toward site-specific reaction. Treatment of breast cancer cells with probe 4 attenuates nuclear hormone receptor activity mediated by retinoic acid, an endogenous client lipid of CRABP2. Our findings demonstrate that arylfluorosulfates can selectively target single iLBPs, making them useful for understanding iLBP function.

Figure 1

Evaluating the proteome reactivity of arylfluorosulfate probes. (a) Alkyne-functionalized arylfluorosulfate probes 1 and 2 and aryl sulfonyl fluoride probe S1. The arylfluorosulfate and aryl sulfonyl fluoride group are shown in red. (b-c) In-gel fluorescence evaluation of the reactivity of probes 1, 2 and S1 in HeLa cells after cell lysis and incorporation of rhodamine-azide using CuAAC. Left panel: in-gel fluorescence, right panel: Coomassie blue staining. (d-e) SILAC ratio plots for proteins identified in experiments comparing cells treated with 1 (d) or 2 (e) versus DMSO (no probe). Proteins with median SILAC ratio ≥ 5 (probe/DMSO) are designated as probe-labeled targets. Ratio ≥ 20 are listed as 20; results display the average of triplicate SILAC experiments performed in HeLa cells. (f) Western blot analysis of probe 1 modified proteins after incorporation of biotin-azide using CuAAC Click and affinity purification using streptavidin agarose beads. Recombinant tag-less human FABP5 serves as the positive control.

Figure 2

(a) Structural alignment of apo CRABP2 (PDBID: 2FS6, green), FABP5 (4LKP, magenta) and FABP4 (3RZY, cyan) in ribbon format by superposition of their protein backbones. Protein side chains of the Arg~Arg~Tyr module are depicted in stick format. (b-c) Tyrosine modification in CRABP2, FABP5 and FABP4 by probe 1, demonstrated by LC-MS/MS analysis. Recombinant proteins (20 µM) were incubated with probe 1 (100 µM) at 25 °C until the modification reached ≥ 80% completion (observed by LC-ESI-MS). Representative fragmentation (MS2) patterns for a peptide containing tyrosine modified with probe 1 is shown for each iLBP analyzed. Identified b and y ions are indicated. A complete list of tryptic peptides identified for each protein, as well as the assignment of the MS2 fragment ion can be found in Supplemental Table S3–S8.

Figure 3

Characterization of the efficient chemoselective modification of CRABP2 by probe 3. (a) Arylfluorosulfate probe 3 bearing a biphenyl moiety. The arylfluorosulfate group is shown in red and the fluorescein moiety is shown in green. (b) pH dependence of modification of CRABP2 (2 µM) by probe 3 (10 µM) in a 10 min reaction period. Top panel: in-gel fluorescence, bottom panel: Coomassie blue staining. (c) Quantification of the relative labeling efficiency from (b) results in an apparent pKa of 7.6 for the Tyr134 phenol group. (d) Comparison of the labeling efficiency for WT, Tyr134Phe, Arg111Leu and Arg132Leu CRABP2 in pH 8.0 and pH 10.4 buffers. Recombinant CRABP2 proteins (2 µM) were incubated with probe 3 (100 µM) at 25 °C for 10 min before quenching the reaction. Top panel: in-gel fluorescence, bottom panel: Coomassie blue staining. (e) Kinetics of probe 3 labeling of CRABP2. Kinetic parameters are indicated. See Experimental Section for definition and calculation of kinetic parameters.

Figure 4

Characterization of the selective modification of overexpressed GFP-CRABP2 by probe 4 in HEK293T cells. (a) Alkyne-functionalized arylfluorosulfate probe 4 bearing a biphenyl moiety. The fluorosulfate group is shown in red. (b) GFP fluorescence of the overexpressed and probe-labeled GFP-CRABP2 and FABP5-GFP analyzed by native PAGE. The modification of GFP-CRABP2 by probe 4 caused a significant band-shift of GFP-CRABP2 to higher molecular weight. The corresponding Coomassie-stained gel is shown in Figure S21. (c) In-gel fluorescence and western blot analysis of the modified GFP-CRABP2 and FABP5-GFP after cell lysis and incorporation of rhodamine-N₃ using CuAAC Click. The corresponding Coomassie-stained gels are shown in Figure S22.

Figure 5

(a) Crystal structure of the probe 4-CRABP2 covalent conjugate at 1.75 Å resolution. The protein moiety is shown in gray and the protein-conjugated probe 4 is shown in pink (only one of two alternative conformations shown for clarity). See Figure S25 for a depiction with both conformations shown. Protein side chains within 5 Å of the probe 4 are depicted in stick format. The side chains of the Arg~Arg~Tyr module are indicated. (b) Analogous view of CRABP2 bound to retinoic acid (RA), shown in yellow. PDB:2FR3.

Figure 6

Inhibition of CRABP2/RARα target gene induction by probe 4. (a) mRNA transcript levels of the RA signaling target gene CRBP1 as measured by qPCR in MCF-7 cells. MCF-7 cells in charcoal-treated FBS were pretreated with 20 µM 4 or DMSO for 4 h and subsequently treated with 100µM RA or EtOH for 24 h. (b-c) mRNA transcript levels of CRABP2 (b) and FABP5 (c) in MCF-7 cells treated under the same conditions as in (a). Error bars show SEM (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001.