3SFM

Novel crystallization conditions for tandem variant R67 DHFR yields wild-type crystal structure

3SFM の概要

• エントリーDOI: 10.2210/pdb3sfm/pdb
• 分子名称: Dihydrofolate reductase type 2, (4R)-2-METHYLPENTANE-2,4-DIOL (3 entities in total)
• 機能のキーワード: oxidoreductase, dihydrofolate reductase, antibiotic resistance, in situ proteolysis, type ii dhfr, sh3, reductase, dhf and nadph-binding
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 7088.03

構造登録者

Yachnin, B.J.,Berghuis, A.M. (登録日: 2011-06-13, 公開日: 2011-11-02, 最終更新日: 2023-09-13)

主引用文献

Yachnin, B.J.,Colin, D.Y.,Volpato, J.P.,Ebert, M.,Pelletier, J.N.,Berghuis, A.M.
Novel crystallization conditions for tandem variant R67 DHFR yield a wild-type crystal structure.
Acta Crystallogr.,Sect.F, 67:1316-1322, 2011

PubMed Abstract: Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66-69 of the first subunit from VQIY to INSF. Using a modified crystallization protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystallized and the structure was solved at 1.4 Å resolution. Surprisingly, only wild-type protomers were incorporated into the crystal. Further experiments demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations.

PubMed: 22102224
DOI: 10.1107/S1744309111030417

実験手法

X-RAY DIFFRACTION (1.4 Å)