1U16

Crystal structure of a duck-delta-crystallin-1 double loop mutant (DLM) in complex with sulfate

1U16 の概要

• エントリーDOI: 10.2210/pdb1u16/pdb
• 関連するPDBエントリー: 1HY0 1HY1 1U15
• 分子名称: Delta crystallin I, SULFATE ION, CHLORIDE ION, ... (5 entities in total)
• 機能のキーワード: eye lens protein, duck-delta-crystallin, argininosuccinate lyase, enzyme mechanism, lyase
• 由来する生物種: Anas platyrhynchos
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 52610.58

構造登録者

Tsai, M.,Sampaleanu, L.M.,Greene, C.,Creagh, L.,Haynes, C.,Howell, P.L. (登録日: 2004-07-14, 公開日: 2004-10-05, 最終更新日: 2023-08-23)

主引用文献

Tsai, M.,Sampaleanu, L.M.,Greene, C.,Creagh, L.,Haynes, C.,Howell, P.L.
A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity.
Biochemistry, 43:11672-11682, 2004

PubMed Abstract: Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

PubMed: 15362851
DOI: 10.1021/bi0489006

実験手法

X-RAY DIFFRACTION (2.2 Å)