Identification of the binding region of the [2Fe-2S] ferredoxin in stearoyl-acyl carrier protein desaturase: insight into the catalytic complex and mechanism of action

Abstract

Stearoyl-acyl carrier protein desaturase (Delta9D) catalyzes the O(2) and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e(-) are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta9D decreased the k(cat)/K(M) for Fd by 4-, 22-, and 2400-fold, respectively, as compared to wt Delta9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K(M) for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K(M) for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta9D and other protein-protein complexes.

Figure 1

(A) Amino acids involved in putative electron transfer pathways to the diiron center (grey spheres) of Δ9D. One proposed pathway, consisting of W62, D228 and H146 (yellow), extends from the surface of Δ9D, and is close to K56, K60 and K230. The other proposed electron transfer pathway consists of W132, F189, Y236, W135 and W139 (blue) and is closest to K41. (B) Surface representation of the Δ9D dimer showing the relative location of the targeted surface lysines (red) in relation to W62 (yellow). The two subunits of Δ9D are shown in different shades of blue.

Figure 2

A summary of the sequence of reactions used for EDC-catalyzed cross-linking. (A) The carboxylate groups from one protein (P1) are activated with EDC and then reacted with NHS to yield a relatively stable succinimide-ester. (B) The NHS-activated carboxylate group on P1 then reacts with an amine group from protein P2 to generate a new peptide bond between proteins P1 and P2 upon displacement of the succinimide group.

Figure 3

Coomassie-stained denaturing electrophoresis gel containing proteins obtained from the indicated cross-linking reactions. The identities of P1 and P2, as defined in Figure 2, are indicated. Lane 1 contains molecular mass standards.

Figure 4

The dependence of v_o for 18:1-ACP formation on the concentration of Fd. (A) wt Δ9D, (○); K41A Δ9D, (□); K60A Δ9D, (x); K56A Δ9D, (◇). (B) K56A/K60A Δ9D, (◆); K230A Δ9D, (●); and K56A/K60A/K230A Δ9D (■). The lines are the results of non-linear least squares fitting of the experimental data using equation 1.

Figure 5

Coomassie-stained denaturing electrophoresis gel showing the influence of 18:0-ACP (abbreviated as ACP to fit the figure) on the cross-linking of Fd to either wt Δ9D (P2 = Δ9D) or K56A/K60A Δ9D (P2 = K56A/K60A Δ9D, abbreviated as Δ9D* to fit in the figure). Lane 1 contains molecular mass standards. Cross-linking of wt Δ9D in the absence (lane 2) or the presence (lane 3) of 18:0-ACP appears similar. Lane 4 shows Δ9D and 18:0-ACP in the presence of EDC, NHS and BME. Cross-linking of K56A/K60A Δ9D is affected by the presence of 18:0-ACP (compare lanes 6 and 7). Activated Fd does not react with 18:0-ACP (lane 5). 18:0-ACP (∼16 kDa) is partially converted to holo-ACP (∼20 kDa) during the electrophoresis.

Figure 6

Predicted sites for protein-protein complex formation between Δ9D and Fd. This orientation allows a view of only one subunit of the Δ9D dimer. (A) Surface representation of Δ9D showing the lysine residues important in Fd binding (red), W62 (yellow), and lysine residues in the ACP binding site (violet). Fd molecules docked at the proposed electron transfer site are shown as green backbone trace; Fd molecules docked in the ACP binding site are shown as red backbone trace. (B) A close-up view of a representative Δ9D-Fd conformation with coloring of the residues as in (A). In this conformation, the [2Fe-2S] cluster of Fd is ∼11 Å from W62.

Figure 7

A model for the tertiary complex of Δ9D. This orientation allows a view of only one subunit of the Δ9D dimer. The Fd and ACP binding sites are separated by ∼50 Å across subunits of the Δ9D dimer. (A) Surface representation of Δ9D with Fd (green) and ACP (red) shown in ribbon representation. Lysine residues involved in forming the Fd binding site are shown in red, W62, D228 and H146 are shown in yellow, and lysine residues in the ACP binding site are shown in pink. The S36 residue of ACP (site of phosphopantetheinylation and acylation) is shown as orange spheres. B) A clipped view representation of Δ9D revealing the position of the channel leading to the active site diiron center (grey spheres). The Fd and ACP are in the same binding sites as show in (A). (C) A close-up view of the region of Δ9D near K230 in the model for the complex of Fd and Δ9D. K230 and E233 are found on the solvent-exposed face of helix-7, which also provides the diiron ligands E229 and H232. E233 is hydrogen bonded to both K230 and R197. It is proposed that these interactions may be perturbed by Fd binding and thus alter the active site in order to facilitate catalysis.