Discovery of posttranslational maturation by self-subunit swapping

Abstract

Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of beta- and alpha-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobalt-containing L-NHase (holo-L-NHase containing Cys-SO(2)(-) and Cys-SO(-) metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt- and oxidized cysteine-containing alpha-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free alpha-subunit of apo-L-NHase with the cobalt-containing cysteine-modified alpha-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named "self-subunit swapping."

Fig. 1.

Expression and purification of L-NHase and holo-αe₂. (A) Genetic organization for the construction of a set of plasmids. The asterisk indicates the mutant nhlA and nhlB genes. (B) SDS/PAGE of a cell-free extract of each R. fascians DSM43985 transformant. (C and D) SDS/PAGE (C) and gel filtration profiles (D) of purified L-NHases and NhlAE. The corresponding genes on the expression plasmid (pREIT series) used for the preparation of L-NHase and NhlAE are shown in italics after each enzyme name.

Fig. 2.

Maturation of apo-L-NHases with holo-αe₂. (A) Elution profiles of apo-α₂β₂ mixed with NhlAE at 0 and 12 h upon gel filtration. (B) Elution profiles of apo-αβ mixed with holo-αe₂ at 0 and 12 h upon gel filtration. (C) SDS/PAGE of the mixture of apo-α₂β₂ and holo-αe₂ (lanes 0 and 12 h), the purified R-α₂β₂ (lane R-α₂β₂), and the αe₂ (lane R-αe₂) purified from the mixture. (D) SDS/PAGE of the mixture of apo-αβ and holo-αe₂ (lanes 0 and 12 h), the purified R-αβ (lane R-αβ), and the αe₂ (lane R-αe₂) purified from the mixture. In A and B, 100 μl of each sample (containing 30 μg of proteins) was applied.

Fig. 3.

MALDI-TOF MS spectra of the metal-binding peptide, EK46, of the apo-α-subunit (A) and holo-α-subunit (B). The mass peak with an m/z value of 5242.36 (A) corresponds to the [M+H]⁺ ion of EK46 with three carboxamidomethylated (CAM−) cysteines (calculated m/z value = 5243.11), and that with an m/z value of 5217.93 (B) corresponds to the [M+H]⁺ ion of EK46 with a Cys-SO₂H and two CAM-cysteines (calculated m/z value = 5218.06). See Discussion in SI Text for more details.

Fig. 4.

Self-subunit swapping maturation of L-NHase. (A) A model of the noncorrin cobalt center of L-NHase. The model is based on all known crystal structures of Co-type NHases (13, 14) and Fe-type NHases (8, 9). Atoms are shown in different colors: pink for Co, black for C, red for O, yellow for S, and blue for N. The salt-bridge networks formed between the modified cysteines and the two arginines are shown as red dotted lines. (B) The proposed heterodimer complexes of holo-L-NHase (holo-αβ) and apo-L-NHase (apo-αβ), and heterotrimer complexes of apo-αe₂ and holo-αe₂. The two cysteines in the apo-α-subunit are denoted as −SH, and the two modified cysteines in the holo-α-subunit as −SO⁻ and SO₂⁻, respectively. The two arginines in the β-subunit are denoted as −Arg⁺. (C) Biosynthesis of apo-L-NHases and holo-αe₂ (Step 1), and self-subunit swapping maturation in vitro (Step 2). (D) Self-subunit swapping maturation in vivo. The cobalt-containing α-subunit is shown in pink.