7LM3

Crystal Structure of Thr316Ala mutant of JAMM domain of S. pombe

7LM3 の概要

• エントリーDOI: 10.2210/pdb7lm3/pdb
• 分子名称: AMSH-like protease sst2, ZINC ION, PHOSPHATE ION (3 entities in total)
• 機能のキーワード: deubiquitinating enzymes, jamm domain, escrt complexes, lus63-linked polyubiquitin chains, metal binding protein
• 由来する生物種: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (Fission yeast)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 44354.99

構造登録者

Shrestha, R.,Das, C. (登録日: 2021-02-05, 公開日: 2021-06-30, 最終更新日: 2023-10-18)

主引用文献

Shrestha, R.,Das, C.
Crystal structure of the Thr316Ala mutant of a yeast JAMM deubiquitinase: implication of active-site loop dynamics in catalysis.
Acta Crystallogr.,Sect.F, 77:163-170, 2021

PubMed Abstract: AMSH, an endosome-associated deubiquitinase (DUB) with a high specificity for Lys63-linked polyubiquitin chains, plays an important role in endosomal-lysosomal sorting and down-regulation of cell-surface receptors. AMSH belongs to the JAMM family of DUBs that contain two insertion segments, Ins-1 and Ins-2, in the catalytic domain relative to the JAMM core found in the archaebacterial AfJAMM. Structural analyses of the AMSH homologs human AMSH-LP and fission yeast Sst2 reveal a flap-like structure formed by Ins-2 near the active site that appears to open and close during its catalytic cycle. A conserved phenylalanine residue of the flap interacts with a conserved aspartate residue of the Ins-1 β-turn to form a closed `lid' over the active site in the substrate-bound state. Analyses of these two residues (Phe403 and Asp315) in Sst2 showed that their interaction plays an important role in controlling the flexibility of Ins-2. The Lys63-linked diubiquitin substrate-bound form of Sst2 showed that the conserved phenylalanine also interacts with Thr316 of Ins-1, which is substituted by tyrosine in other AMSH orthologs. Although Thr316 makes no direct interaction with the substrate, its mutation to alanine resulted in a significant loss of activity. In order to understand the contribution of Thr316 to catalysis, the crystal structure of this mutant was determined. In spite of the effect of the mutation on catalytic activity, the structure of the Sst2 Thr316Ala mutant did not reveal significant changes in either the overall structure or the active-site arrangement relative to the wild type. The Phe403-Thr316 van der Waals interaction is impaired by the Thr316Ala mutation, abrogating the adoption of the closed active-site conformation required for catalysis. Since van der Waals interactions with phenylalanine are conserved across substrate-bound forms of AMSH-LP and Sst2, these interactions may be critical for loop immobilization and the positioning of the isopeptide bond of Lys63-linked polyubiquitin-chain substrates.

PubMed: 34100774
DOI: 10.1107/S2053230X21005124

実験手法

X-RAY DIFFRACTION (2.7 Å)