Three-stage assembly of the cysteine synthase complex from Escherichia coli

Abstract

Control of sulfur metabolism in plants and bacteria is linked, in significant measure, to the behavior of the cysteine synthase complex (CSC). The complex is comprised of the two enzymes that catalyze the final steps in cysteine biosynthesis: serine O-acetyltransferase (SAT, EC 2.3.1.30), which produces O-acetyl-L-serine, and O-acetyl-L-serine sulfhydrylase (OASS, EC 2.5.1.47), which converts it to cysteine. SAT (a dimer of homotrimers) binds a maximum of two molecules of OASS (a dimer) in an interaction believed to involve docking of the C terminus from a protomer in an SAT trimer into an OASS active site. This interaction inactivates OASS catalysis and prevents further binding to the trimer; thus, the system exhibits a contact-induced inactivation of half of each biomolecule. To better understand the dynamics and energetics that underlie formation of the CSC, the interactions of OASS and SAT from Escherichia coli were studied at equilibrium and in the pre-steady state. Using an experimental strategy that initiates dissociation of the CSC at different points in the CSC-forming reaction, three stable forms of the complex were identified. Comparison of the binding behaviors of SAT and its C-terminal peptide supports a mechanism in which SAT interacts with OASS in a non-allosteric interaction involving its C terminus. This early docking event appears to fasten the proteins in close proximity and thus prepares the system to engage in a series of subsequent, energetically favorable isomerizations that inactivate OASS and produce the fully isomerized CSC.

FIGURE 1.

The fluorescence of OASS-bound PLP. A, emission spectra of different OASS·PLP complexes. Spectra are given for OASS in complex with SAT (black), peptide (green), and free enzyme (red) and following reaction with OAS (blue). OASS_dimer was 0.50 μm in all cases. The ligand concentrations were: SAT_trimer (1.0 μm), or OAS (1.0 mm), or peptide (50 μm), or none. Solutions were buffered with Hepes/K⁺ (50 mm, pH 7.0), and spectra were acquired at T = 25 ± 2 °C. Samples were excited at λ = 414 nm. B, the structure of OASS bound to the C-terminal peptide of SAT. The figure was prepared using PyMOL, and the color scheme is as follows: peptide (blue), PLP (by atom), and OASS (wheat).

FIGURE 2.

Isomerization during cysteine synthase formation. A, the OAS-induced dissociation of the CSC. OAS was added to an equilibrated solution of CSC, and the ensuing dissociation reaction was allowed to reach equilibrium. The normalized fluorescence (I/I₀) reports the CSC concentration and indicates complete dissociation of the complex at OAS concentrations above 50 μm. The conditions were as follows: OASS_dimer (1.0 μm), SAT_trimer (1.0 μm), Hepes/K⁺ (50 mm, pH 7.0), and OAS concentrations are indicated in the figure; T = 25 ± 1 °C. B, the CSC dissociation progress curve. Dissociation was initiated by addition of OAS (0.10 mm final, from 60 mm stock) to a solution containing CSC (0.25 μm) that was formed by equilibration of OASS_dimer (0.25 μm) and SAT_trimer (2.0 μm) in Hepes/K⁺ (50 mm, pH 7.0); T = 25 ± 2 °C. Dissociation was monitored via fluorescence (λ_ex 414 nm, λ_em 510 nm). The solid line represents the best-fit of the data to a single exponential decay, where k_off = 0.0008 ± 0.0001 s⁻¹. C, association of the CSC. The reaction was initiated by mixing a solution containing OASS_dimer (0.10 μm) with an equal volume of a solution containing SAT_trimer (8.0 μm). The solutions were equilibrated prior to mixing at 25 ± 2 °C in Hepes/K⁺ (50 mm, pH 7.0). Reactions were monitored by following fluorescence intensity (λ_ex 414 nm, a cut-off filter was used to detected emitted light above 455 nm). The progress curve shown is the average of eight repetitions. The curve passing through the data represents the best-fit of a single-exponential model. D, k_obs versus [SAT] for CS formation. k_obs values were obtained from progress curves generated over a series of SAT concentrations using the methods and conditions described in B. The reactions were performed in triplicate and were pseudo first order with respect to OASS in all cases. k_on and k_off were obtained from the best fit straight line to the k_obs versus [SAT] data; k_on = 8.1 ± 0.2 ·10⁶ m⁻¹s⁻¹, k_off = 6.1 ± 0.9 s⁻¹.

FIGURE 3.

Reaction-stage-dependent dissociation of the CS complex reveals an additional isomerization. A, dissociation progress curves. Binding reactions were initiated by rapidly mixing equal volumes of OASS_dimer (0.40 μm) and SAT_trimer (16.0 μm). The reactions were allowed to proceed over a series of time intervals, the delay times, before the complex was irreversibly dissociated by mixing rapidly with an equal volume of OAS (0.40 mm). The dissociation reactions were monitored via fluorescence changes (λ_ex 414 nm, λ_em 510 nm). The white lines represent the behavior predicted by double-exponential best fits of the data. All solutions were buffered using Hepes/K⁺ (50 mm, pH 7.0) and equilibrated at T = 25 ± 1 °C prior to mixing. B, exponential decay of the fast-phase amplitude. The fast-phase amplitudes predicted by double-exponential, best fits of the individual dissociation reactions are plotted versus the delay (or reaction) times to reveal the way in which the amplitude decays over time. The amplitude data were well fit using a single-exponential model, which yielded a best-fit rate constant of 4.1 ± 0.3 s⁻¹.

FIGURE 4.

The CSC assembly mechanism. The complexes and their associated rate constants are shown. Rate constants are labeled with their experimentally determined values. Long arrows indicate regions of the reaction that are associated with composite rate constants. Isomerized forms are indicated with single and double prime symbols.

FIGURE 5.

The interaction of peptide (P₁₀) with OASS. A, the dissociation constant. Peptide binding to OASS was monitored via fluorescence changes at 510 nm (λ_ex 414 nm). Titrations were carried out in Hepes/K⁺ (50 mm, pH 7.0) at 25 ± 2 °C. The concentration of OASS_dimer was 0.50 μm. B, rate constants. Binding was initiated by rapidly mixing (1:1) OASS_dimer (6.25 nm, final) with P₁₀ (at the concentrations indicated). Solutions were buffered using Hepes/K⁺ (50 mm, pH 7.0) and equilibrated at 25 ± 2 °C prior to mixing. Reaction progress was monitored using fluorescence (λ_ex 414 nm, λ_em > 455 nm). k_obs values were determined in triplicate: each value was obtained by fitting the average of ten progress curves to a single-exponential decay. k_on (7.7 × 10⁶ m⁻¹ s⁻¹) and k_off (3.6 s⁻¹) were obtained by fitting the k_obs versus [P₁₀] data using the equation: k_obs = k_on · [pep] + k_off.

FIGURE 6.

SAT binding prevents access to the unoccupied subunit of the OASS dimer. A, the stoichiometries of the peptide·OASS and SAT·OASS complexes. Equilibrium-binding titrations were carried out in Hepes/K⁺ (50 mm, pH 7.0) at 25 ± 2 °C, and binding was monitored via fluorescence changes (λ_ex 414 nm, λ_em 510 nm). The OASS dimer concentrations used in the SAT and peptide titrations were 1.25 μm and 80 μm, respectively. B, SAT binding prevents P₁₀ binding at the unoccupied site of OASS. OASS_dimer (1.0 μm) was titrated to saturation with SAT and peptide was then added at the concentrations indicated. Conditions were otherwise identical to those described in A.