1SY3

1.00 A Crystal Structure of D30N Mutant of Nitrophorin 4 from Rhodnius Prolixus Complexed with Nitric Oxide

1SY3 の概要

• エントリーDOI: 10.2210/pdb1sy3/pdb
• 関連するPDBエントリー: 1KOI 1SXU 1SXW 1SXX 1SXY 1SY0 1SY1 1SY2
• 分子名称: Nitrophorin 4, PHOSPHATE ION, PROTOPORPHYRIN IX CONTAINING FE, ... (5 entities in total)
• 機能のキーワード: lipocalin, beta barrel, ferrous heme, nitric oxide, transport protein
• 由来する生物種: Rhodnius prolixus
• 細胞内の位置: Secreted: Q94734
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 21033.14

構造登録者

Maes, E.M.,Weichsel, A.,Andersen, J.F.,Shepley, D.,Montfort, W.R. (登録日: 2004-03-31, 公開日: 2004-06-08, 最終更新日: 2023-08-23)

主引用文献

Maes, E.M.,Weichsel, A.,Andersen, J.F.,Shepley, D.,Montfort, W.R.
Role of binding site loops in controlling nitric oxide release: structure and kinetics of mutant forms of nitrophorin 4
Biochemistry, 43:6679-6690, 2004

PubMed Abstract: Nitrophorins are ferric heme proteins that transport nitric oxide (NO) from blood-sucking insects to victims. NO binding is tighter at lower pH values, as found in the insect salivary gland, and weaker at the pH of the victim's tissue, facilitating NO release and subsequent vasodilation. Previous structural analyses of nitrophorin 4 (NP4) from Rhodnius prolixus revealed a substantial NO-induced conformational change involving the A-B and G-H loops, which rearrange to desolvate the distal pocket and pack nonpolar residues against the heme-ligated NO. Previous kinetic analyses revealed a slow, biphasic, and pH-dependent NO release, which was proposed to be associated with loop movements. In this study, we created NP4 mutants D30A and D30N (A-B loop), D129A/L130A (G-H loop), and T121V (distal pocket). Eight crystal structures were determined, including complexes with NO, NH(3), and imidazole, to resolutions as high as 1.0 A. The NO-induced conformational change is largely abolished in the loop mutants, but retained in T121V. Kinetic analyses using stopped-flow spectroscopy revealed the pH dependence for NO release is eliminated for D129A/L130A, considerably reduced for D30A and D30N, but retained for T121V. NO association rates were increased 2-5-fold for T121V, but were unchanged in the loop mutants. Taken together, our findings demonstrate that the pH dependency for NO release is linked to loop dynamics and that solvent reorganization is apparently rate-limiting for formation of the initial iron-nitrosyl bond. Interestingly, the multiphasic kinetic behavior of rNPs was not affected by mutations, and its cause remains unclear.

PubMed: 15157102
DOI: 10.1021/bi049748a

実験手法

X-RAY DIFFRACTION (1 Å)