Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases

Abstract

We describe a general strategy for selective chemical labeling of individual adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using active site-directed proteomic probes coupled to the 5'-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.

Figure 1

Methods for proteomic analysis of A domains in NRPS biosynthetic enzymes using active site-directed proteomic probes for A domains. Modules are comprised of thiolation (T), adenylation (A) [Af: l-Phe; Ap: l-Pro; Av: l-Val; Ao: l-Orn specific A domains[, epimerization (E), condensation (C), and thioesterase (TE) domains. In a gel-based analysis, recombinant NRPS enzymes or proteomes treated with individual probes are incubated with a rhodamine (Rh)-azide reporter tag under click chemistry (CC) conditions and separated by SDS-PAGE, and labeled A domains in NRPS enzymes are visualized by in-gel fluorescence imaging.

Figure 2

(a) Biosynthetic pathway of the gramicidine S. GrsA is composed of A₁ (l-Phe), T, and E domains. In contrast, GrsB consists of four NRPS modules, C-A₂ (l-Pro)-T-C-A₃ (l-Val)-T-C-A₄ (l-Orn)-T-C-A₅ (l-Leu)-T-TE. (b) The adenylation reaction in NRPS. (c) Structures of active site-directed proteomic probes and inhibitors for A domains described in this study.

Figure 2

(a) Biosynthetic pathway of the gramicidine S. GrsA is composed of A₁ (l-Phe), T, and E domains. In contrast, GrsB consists of four NRPS modules, C-A₂ (l-Pro)-T-C-A₃ (l-Val)-T-C-A₄ (l-Orn)-T-C-A₅ (l-Leu)-T-TE. (b) The adenylation reaction in NRPS. (c) Structures of active site-directed proteomic probes and inhibitors for A domains described in this study.

Figure 3

Labeling of recombinant holo-GrsA and holo-TycB₁ with probes 1 and 2. (a) Labeling of GrsA and TycB1 and competitive inhibition studies with excess inhibitors 4 and 5. GrsA (1 µM) and TycB1 (1 µM) were individually pre-incubated in either the absence or presence of 100 µM of inhibitors 4 and 5 and treated with 1 µM of the individual probes 1 and 2. (b) Ultraviolet photolysis time course studies of the labeling of GrsA with probe 1 (left) and TycB1 with probe 2 (right). SDS-PAGE analysis denoting the labeling of 1 µM of GrsA and TycB1 with 1 µM probes 1 and 2, respectively. (c, d) Limit of detection of GrsA and TycB1 labeling. GrsA (0.125–62.5 nM) and TycB1 (0.125–62.5 nM) were individually incubated with probes 1 and 2. (e, f) Labeling specificity of probes 1 and 2. GrsA (1 µM), TycB1 (1 µM), AusA₁ (1 µM), and BSA (1 µM) were individually treated with 1 µM of probes 1 and 2 in either the absence or presence of 100 µM of the inhibitors 4, 5, and 6. For each panel, Φ depicts the fluorescence observed with λ_ex = 532 nm and λ_em = 580 nm, and Σ displays the total protein content by staining with Coomassie Blue (a, b, e, and f) or a silver staining method (c and d). Full gels (Figures S3) and experimental procedures are provided in the SI.

Figure 4

Proteomic applications of active site-directed proteomic probes 1–3 for A domains. (a) Labeling of endogenous GrsA in an A. migulanus ATCC 9999 cellular lysate by probe 1. The A. migulanus ATCC 9999 lysate (1.5 mg/mL) was treated with 1 µM 1 in either the absence or presence of 100 µM 4. (b) Labeling of endogenous GrsB in the A. migulanus DSM 5759 cellular lysate by probes 2 and 3. The A. migulanus DSM 5759 lysate (1.5 mg/mL) was individually treated with 100 µM 5 and 7 before the addition of individual members of 1 µM probes 2 and 3. (d) Individual labeling of A domains and profiling of A domain functions using a combination of probes 1–3 and inhibitors 4–8. In order to investigate GrsA labeling, the A. migulanus ATCC 9999 lysate (1.5 mg/mL) was preincubated with individual members of inhibitors 4–8 (100 µM) before the addition of 1 µM of probe 1. To evaluate the labeling of GrsB, the A. migulanus DSM 5759 lysate (1.5 mg/mL) was individually treated with 100 µM of inhibitors 4–8 before the addition of individual members of 1 µM probes 2 and 3. For each panel, Φ depicts the fluorescence observed with λ_ex = 532 nm and λ_em = 580 nm, and Σ displays the total protein content by staining with Coomassie Blue. Full gels (Figure S4) and experimental procedures are provided in the SI.