Trapping the Complex Molecular Machinery of Polyketide and Fatty Acid Synthases with Tunable Silylcyanohydrin Crosslinkers

Abstract

Many families of natural products are synthesized by large multidomain biological machines commonly referred to as megasynthases. While the advance of mechanism-based tools has opened new windows into the structural features within the protein-protein interfaces guiding carrier protein dependent enzymes, there is an immediate need for tools that can be engaged to link co-translated domains in a site-selective manner. Now, the use of silylcyanohydrins is demonstrated in a two-step, two-site selective crosslinking for the trapping of carrier-protein interactions within megasynthases. This advance provides a new tool to trap intermediate states within multimodular systems, a key step toward understanding the specificities within fatty acid (FAS) and polyketide (PKS) synthases.

Keywords: biosynthesis; natural products; protein crosslinking; protein labeling; synthases.

Figure 1.

Schematic representation of the mycocerosic acid synthase (MAS). a) Structure of mycocerosic acid. b) Steps involved in ketide processing in MAS. The acyl carrier protein (ACP, blue) interacts with five different domains sequentially beginning with the acyltransferase (AT, red) and ending with the enoyl reductase (ER, dark green) as different domains come in contact with the ACP (circles). c) An expansion of the MAS system depicting the AT, ACP, ketosynthase (KS, orange), ketoreductase (KR, yellow), dehydratase (DH, light green), and ER.

Figure 2.

Structures of crosslinking agents: sulfone 1, dimethylethyl (DMES)-cyanohydrin 3, triethylsilyl-(TES)-cyanohydrin 4, and t-butyldimethylsilyl-(TBS)-cyanohydrin 5. Inhibitor 2, sulfone 6, ketone 7, cyanohydrin 8, trimethylsilyl-(TMS)-cyanohydrin 9, DMES-cyanohydrin 10, and TES-cyanohydrin 11 were used for optimization. Studies with the glycine-linked equivalent of 6 (see S23 in Fig. S2) were conducted to evaluate the role of the linker.

Figure 3.

Tunable fluorescent labeling of DH domains. a) A SDS-PAGE gel depicting the fluorescence (top) and total protein (bottom) from the incubation of 2 µM aliquot of FabA with 20 µM 6 at 37 °C, 2 µM 7 at 25 °C, 2 µM 8 at 25 °C, 2 µM 9 at 25 °C, 2 µM 10 at 25 °C or 2 µM 11 at 25 °C in 100 mM Tris•HCl pH 8.0 for 12 h. b) A SDS-PAGE gel depicting the fluorescence (top) and total protein (bottom) from the incubation of 2 µM aliquot of FabA with 2 µM 7, 2 µM 9, 2 µM 10 or 2 µM 11 at 25 °C for 12 h in 100 mM Tris•HCl pH 8.0 in the presence (+) or absence (–) of 100 mM KF. c) SDS-PAGE gels depicting the effects of pH on the incubation of 2 µM FabA with 2 µM 7, 2 µM 8, or 2 µM 10 in 100 mM Tris•HCl at 25 °C for 12 h in the absence (–) or presence (+) of 100 mM KF. d) A SDS-PAGE gel depicting the fluorescence (left) and total protein (right) from the incubation of 2 µM of each protein with 2 µM 7 or 2 µM 10 in 100 mM Tris•HCl pH 7.0 at 25 °C for 12 h in the absence (–) or presence (+) of 100 mM KF. e) A SDS-PAGE gel depicting the fluorescence from the labeling of a 1.0 mg/mL aliquot of lysate from K12 E. coli, which was spiked with 2 µM FabA, and then treated with 20 µM 6 at 37 °C, 2 µM 7 at 25 °C, or 2 µM 10 at 25 °C in 100 mM Tris•HCl 150 mM NaCl pH 8.0 for 12 h in the absence (–) or presence (+) of 100 mM KF. f) Total protein stained gel in e). Protein bands are indicated by b1=FabA; b2=FabF; b3=FabD; b4=FabI; b5=FabG; and b6=AcpP. Comparison between reactions with and without 200 µM inhibitor 2 confirmed the site selectivity of the labeling. Supporting Fig. S7 provides full-scale gel images. Gel shifts were observed when appending 2, 7, 9 and 10 to FabA.

Figure 4.

Crosslinking studies. a) A SDS PAGE gel depicting 10 µM FabA that was incubated with 10 µM crypto-AcpP loaded with 1, 3, 4 or 5 in the absence (–) or presence (+) of 100 mM KF 50 mM Tris•HCl pH 8.5, 150 mM NaCl and 10% glycerol at 37 °C for 16 h. b) A SDS PAGE gel depicting 10 µM FabA that was incubated with 10 µM crypto-AcpP, 10 µM crypto-ACP4, 10 µM crypto-act-ACP or 10 µM crypto-PltL bearing either 1 or 4 in the absence (–) or presence (+) of 100 mM KF in 50 mM Tris·HCl pH 8.5, 150 mM NaCl and 10% glycerol at 37 °C for 24 h. c) A SDS PAGE gel depicting 10 µM DH4 that was incubated with 10 µM crypto-AcpP, 10 µM crypto-ACP4, 10 µM crypto-act-ACP or 10 µM crypto-PltL bearing either 1 or 4 in the absence (–) or presence (+) of 100 mM KF in 50 mM Tris•HCl pH 8.5, 150 mM NaCl and 10% glycerol at 37 °C for 24 h. d) SDS PAGE gel depicting 1 μM apo- or crypto-MAS loaded with 1, 3, 4 or 5 in the absence (−) or presence (+) of 100 mM KF 50 mM Tris•HCl pH 7.0, 150 mM NaCl and 10% glycerol at 25 °C for 12 h. Fig. S8 provides further support the intramolecular crosslinked species at 280 kDa. Bands are given by: b1=FabA=AcpP; b2=holo-AcpP; b3=FabA=ACP4; b4=FabA=act-ACP or FabA=PltL; b5=DH4=ACP4; b6=DH4=AcpP; b7 = DH4=act-ACP or DH4=PltL; b8 = apo- or crypto-MAS; or b9 = crosslinked crypto-MAS Supporting Fig. S7 provides full-scale gel images.

Scheme 1.

Silylcyanohydrin crosslinking. a) A view of a synthase undergoing an ACP•DH interaction, with a zoom onto the protein • protein interface (circle) and substrate binding pocket (square). b) Two-step silylcyanohydrin caged DH crosslinking. One pot loading of 3-5 onto the ACP results in crypto-ACP 12, which can be purified and stored. The crosslinking reaction is induced by the addition of KF to a mixture of 12 and a DH. After deprotection, 13 binds to the DH and inserts its cargo into the DH active site, as shown in 14. HCN is eliminated, and the resulting complex 15 can covalently modify proximal His residues (Supporting Fig. S1).^[15] Alternatively, the silylcyanohydrin in 12 can bind to the DH, and the subsequent steps could occur in the DH active site.