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.

中文翻译：

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酵母 JAMM 去泛素酶 Thr316Ala 突变体的晶体结构：活性位点环动力学在催化中的意义

AMSH 是一种内体相关去泛素酶 (DUB)，对 Lys63 连接的多聚泛素链具有高度特异性，在内体-溶酶体分选和细胞表面受体的下调中发挥重要作用。AMSH 属于 DUB 的 JAMM 家族，其在与古细菌 AfJAMM 中发现的 JAMM 核心相关的催化结构域中包含两个插入片段 Ins-1 和 Ins-2。对 AMSH 同源物人 AMSH-LP 和裂殖酵母 Sst2 的结构分析揭示了 Ins-2 在活性位点附近形成的瓣状结构，该结构在其催化循环过程中似乎打开和关闭。皮瓣的保守苯丙氨酸残基与 Ins-1 β-转角的保守天冬氨酸残基相互作用，在底物结合状态的活性位点上形成封闭的“盖子”。对Sst2中这两个残基（Phe403和Asp315）的分析表明，它们的相互作用在控制Ins-2的灵活性中发挥着重要作用。Sst2 的 Lys63 连接的双泛素底物结合形式表明，保守的苯丙氨酸也与 Ins-1 的 Thr316 相互作用，该 Thr316 在其他 AMSH 直系同源物中被酪氨酸取代。尽管 Thr316 不与底物直接相互作用，但其突变为丙氨酸导致活性显着丧失。为了了解 Thr316 对催化的贡献，确定了该突变体的晶体结构。尽管突变对催化活性有影响，但Sst2 Thr316Ala突变体的结构并未显示出相对于野生型的整体结构或活性位点排列的显着变化。Phe403-Thr316 范德华相互作用因 Thr316Ala 突变而受损，从而取消了催化所需的封闭活性位点构象的采用。由于与苯丙氨酸的范德华相互作用在 AMSH-LP 和 Sst2 的底物结合形式中是保守的，因此这些相互作用可能对于 Lys63 连接的多聚泛素链底物的环固定和异肽键的定位至关重要。