Identifying esterase and lipase activity in proteins of unknown function
Identifying esterase and lipase activity in proteins of unknown function
复制标题
鉴定未知功能蛋白质中的酯酶和脂肪酶活性
DOI：
10.1016/j.bpj.2022.11.2580
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发表时间：
2023
影响因子：
3.4
通讯作者：
Cup, Theodore
中科院分区：
文献类型：
--
作者：
Koeppe, Julia R.;Serrano, Christopher;Thompson, Sylvia;Holliday, Gregg;Singh, Veronica;Cup, Theodore
患者突变已被确定在整个动力蛋白相关蛋白1（Drp 1），线粒体分裂的关键介质。这些突变通常会影响儿童，并经常导致严重的神经缺陷，在某些情况下甚至导致死亡。以前，基于与相似位点的合成突变的比较，推测了导致患者表型的潜在功能缺陷。Drp 1由四个结构域组成，包括调节GTP结合和水解的GTP酶域（G-结构域）和介导Drp 1自组装的中间结构域（MD）。我们分析了六个突变，四个在MD，两个在G结构域。所有研究的MD突变体被发现在溶液中是二聚体限制的，并且发现这些MD突变体中的三个在自组装中受损。然而，其中一个MD突变体保留寡聚化能力，尽管位于该自组装区域内。此外，该突变体保留其与预形成的脂质模板相互作用的能力，但不能重塑大的单层囊泡（LUV）。这表明单独的Drp 1二聚体足以在脂质模板上形成螺旋聚合物。在两种G结构域突变之间也观察到差异。其中一个突变体位于对功能性水解至关重要的核苷酸结合环中。该突变体表现出受损的GTP水解，但仍然可以在溶液中和与脂质模板寡聚化。相反，另一个G结构域突变位于相对远离核苷酸结合位点。这种突变体不良微管LUV，突出G-结构域的相互作用，在驱动膜曲率的作用。总的来说，由Drp 1突变引起的功能缺陷是高度可变的，即使突变发生在相同的功能结构域内。这项研究提供了一个框架，用于表征额外的Drp 1突变，以全面了解这种必需蛋白质中的功能位点。
Patient mutations have been identified throughout dynamin-related protein 1 (Drp1), the key mediator of mitochondrial fission. These mutations generally impact children and often result in severe neurological defects and, in some cases, death. Previously, the underlying functional defect leading to patient phenotypes has been speculated based on comparisons with synthetic mutations at similar sites. Drp1 is comprised by four domains, including a GTPase domain (G-domain) that regulates GTP binding and hydrolysis and a middle domain (MD) that mediates Drp1 self-assembly. We analyzed six mutations, four in the MD and two in the G-domain. All the MD mutants studied were found to be dimerlimited in solution, and three of these MD mutants were found to be impaired in self-assembly. However, one of the MD mutants retains oligomerization capability despite being located within this self-assembly region. Further, this mutant retains its ability to interact with a pre-formed lipid template but is unable to reshape large, unilamellar vesicles (LUVs). This indicates that Drp1 dimers alone are sufficient to form a helical polymer on lipid template. Differences were also observed between the two G-domain mutations. One of the mutants is in a nucleotide-binding loop that is critical for functional hydrolysis. This mutant exhibited impaired GTP hydrolysis but can still oligomerize in solution and with a lipid template. In contrast, the other G-domain mutation is located relatively distant from the nucleotide binding site. This mutant poorly tubulates LUVs, highlighting the role of G-domain interactions in driving membrane curvature. Overall, the functional defects caused by mutations in Drp1 are highly variable even when the mutations occur within the same functional domain. This study provides a framework for characterizing additional Drp1 mutations to provide a comprehensive understanding of functional sites within this essential protein.