PPubMed15 May 2010Cited 23×

Structure of <i> Arabidopsis </i> chloroplastic monothiol glutaredoxin AtGRXcp Monothiol glutaredoxin

Lenong Li, Ninghui Cheng, Kendal D. Hirschi, Xiaoqiang Wang

Abstract

The structure of Arabidopsis monothiol glutaredoxin AtGRXcp has been determined and reveals unique structural features of monothiol glutaredoxins, key residues for their interaction with glutathione and structural determinants for their distinct biochemical properties.

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The 2.4 Å crystal structure of Arabidopsis monothiol glutaredoxin AtGRXcp reveals a unique glutathione‑binding groove and a five‑residue loop that likely dictate its distinct biochemical behavior.

What’s new

i.First crystal structure of a plant CGFS‑type monothiol glutaredoxin (AtGRXcp).
ii.Identification of a distinct glutathione‑binding groove with key residues Lys89, Arg126, Asp152.
iii.Discovery of a unique five‑amino‑acid loop adjacent to the active‑site motif as a structural determinant.
iv.Modeling of a potential [2Fe‑2S] cluster binding mode and observation of an intermolecular Cys172 disulfide bond.

How they did it

AtGRXcp (without its chloroplast signal peptide) was cloned into pET‑41a, expressed in E. coli, purified by affinity and gel‑filtration, crystallized by hanging‑drop vapor diffusion, and its structure solved to 2.4 Å by X‑ray diffraction and molecular replacement, followed by refinement and molecular docking of glutathione.

What they found

Structure resolved at 2.4 Å resolution with R = 19.2 % and Rfree = 22.6 %.
One protein molecule in the asymmetric unit; residues 65‑173 modeled.
RMSD of 1.3 Å to E. coli Grx4 (36 % sequence identity) and 1.8 Å to human Grx2.
Glutathione‑binding groove measured 11‑14 Å wide and 16‑19 Å long.
Five‑residue loop (Thr91‑Arg92‑Asp93‑Phe94‑Pro95) identified adjacent to Cys97.

Caveats

✕Crystals of the AtGRXcp‑glutathione complex could not be obtained, so binding mode is based on docking.
✕Active‑site motif shows higher flexibility, limiting certainty about its exact conformation.
✕Functional role of the five‑residue loop is inferred from structure, not experimentally validated.
✕Potential [2Fe‑2S] cluster binding is modeled, not directly observed.

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