4ZM9

Crystal structure of circularly permuted human asparaginase-like protein 1

4ZM9 の概要

• エントリーDOI: 10.2210/pdb4zm9/pdb
• 関連するPDBエントリー: 4ET0
• 分子名称: Isoaspartyl peptidase/L-asparaginase, SODIUM ION, GLYCINE, ... (7 entities in total)
• 機能のキーワード: circularly permutated, human asparaginase, hydrolase
• 由来する生物種: Homo sapiens (Human)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 136997.90

構造登録者

Li, W.Z.,Zhang, Y. (登録日: 2015-05-02, 公開日: 2016-02-17, 最終更新日: 2024-03-06)

主引用文献

Li, W.,Irani, S.,Crutchfield, A.,Hodge, K.,Matthews, W.,Patel, P.,Zhang, Y.J.,Stone, E.
Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages.
Biochemistry, 55:960-969, 2016

PubMed Abstract: The human asparaginase-like protein 1 (hASRGL1) is a member of the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds into an αβ-βα sandwich fold homodimer that cleaves its own peptide backbone at the G167-T168 bond, resulting in the active form of the enzyme. However, biophysical studies of hASRGL1 are difficult because of the curious fact that intramolecular cleavage of the G167-T168 peptide bond reaches only ≤50% completion. We capitalized upon our previous observation that intramolecular processing increases thermostability and developed a differential scanning fluorimetry assay that allowed direct detection of distinct processing intermediates for the first time. A kinetic analysis of these intermediates revealed that cleavage of one subunit of the hASRGL1 subunit drastically reduces the processing rate of the adjacent monomer, and a mutagenesis study showed that stabilization of the dimer interface plays a critical role in this process. We also report a comprehensive analysis of conserved active site residues and delineate their relative roles in autoprocessing and substrate hydrolysis. In addition to glycine, which was previously reported to selectively accelerate hASRGL1 cleavage, we identified several novel small molecule activators that also promote intramolecular processing. The structure-activity analysis supports the hypothesis that multiple negatively charged small molecules interact within the active site of hASRGL1 to act as a base in promoting cleavage. Overall, our investigation provides a mechanistic understanding of the maturation process of this Ntn hydrolase family member.

PubMed: 26780688
DOI: 10.1021/acs.biochem.5b01157

実験手法

X-RAY DIFFRACTION (2.51 Å)