NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4
NMR and MD simulations reveal the impact of the V23D mutation on the function of yeast oligosaccharyltransferase subunit Ost4
复制标题
NMR 和 MD 模拟揭示了 V23D 突变对酵母寡糖转移酶亚基 Ost4 功能的影响
DOI：
10.1093/glycob/cwab002
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发表时间：
2021
期刊：
影响因子：
4.3
通讯作者：
Mohanty, Smita
中科院分区：
文献类型：
--
作者：
Chaudhary, Bharat P;Zoetewey, David L;McCullagh, Martin J;Mohanty, Smita
天冬酰胺连接的糖基化，也称为N-连接的糖基化，是真核生物和一些原核生物中必需的和高度保守的共翻译和翻译后蛋白质修饰。在该反应的中心步骤中，碳水化合物部分从脂质连接的供体转移到新生蛋白质中共有天冬酰胺的侧链，因为它在核糖体处合成。寡糖基转移酶（OST）功能的完全丧失在真核生物中是致命的。该反应由位于内质网中的膜相关多亚基酶OST进行。最小的亚基Ost 4包含一个跨膜螺旋，这对于维持OST的稳定性和活性至关重要。从Met 18到Ile 24的任何残基的突变都会使酶复合物不稳定，影响其活性。在这里，我们报告解决方案的核磁共振结构和分子动力学（MD）模拟Ost 4和Ost 4V 23 D胶束。我们的研究表明，虽然点突变不影响蛋白质的结构，但它会影响其在膜模拟环境中的位置和溶剂暴露。此外，我们对含有WT或V23 D突变体的膜结合OST复合物的MD模拟证明了Ost 4V 23 D与Stt 3的跨膜TM 12和TM 13之间的大多数疏水螺旋-螺旋相互作用的破坏。Ost 4V 23 D从OST复合物的这种脱离导致D23残基在由这些相互作用产生的疏水口袋中暴露于溶剂。本研究不仅解决了酵母Ost 4亚基及其突变体的结构问题，而且为OST复合物的失稳和OST活性的降低提供了依据。
Asparagine-linked glycosylation, also known as N-linked glycosylation, is an essential and highly conserved co- and post-translational protein modification in eukaryotes and some prokaryotes. In the central step of this reaction, a carbohydrate moiety is transferred from a lipid-linked donor to the side-chain of a consensus asparagine in a nascent protein as it is synthesized at the ribosome. Complete loss of oligosaccharyltransferase (OST) function is lethal in eukaryotes. This reaction is carried out by a membrane-associated multisubunit enzyme, OST, localized in the endoplasmic reticulum. The smallest subunit, Ost4, contains a single membrane-spanning helix that is critical for maintaining the stability and activity of OST. Mutation of any residue from Met18to Ile24of Ost4 destabilizes the enzyme complex, affecting its activity. Here, we report solution nuclear magnetic resonance structures and molecular dynamics (MD) simulations of Ost4 and Ost4V23D in micelles. Our studies revealed that while the point mutation did not impact the structure of the protein, it affected its position and solvent exposure in the membrane mimetic environment. Furthermore, our MD simulations of the membrane-bound OST complex containing either WT or V23D mutant demonstrated disruption of most hydrophobic helix–helix interactions between Ost4V23D and transmembrane TM12 and TM13 of Stt3. This disengagement of Ost4V23D from the OST complex led to solvent exposure of the D23 residue in the hydrophobic pocket created by these interactions. Our study not only solves the structures of yeast Ost4 subunit and its mutant but also provides a basis for the destabilization of the OST complex and reduced OST activity.