Core structures of ubiquitin dictate its dynamics and function
Core structures of ubiquitin dictate its dynamics and function
复制标题
泛素的核心结构决定其动力学和功能
DOI：
10.1016/j.jmb.2013.12.008
复制
发表时间：
2014
影响因子：
5.6
通讯作者：
S.
中科院分区：
文献类型：
--
作者：
Fukai;S.
在溶剂不可接近的核心位置的突变可能会影响蛋白质的功能，通过改变蛋白质动力学。然而，这样的突变也可能改变蛋白质的整体结构和/或热力学稳定性，因此，它们的影响在许多情况下难以解释。在这一期的《分子生物学杂志》上，Fushman、Bolon和他们的同事挑战了将泛素作为参考分子研究的困难。泛素是从酵母到哺乳动物高度保守的76个残基的蛋白质。泛素的折叠由一个五链β折叠、一个短的310螺旋和一个α螺旋组成[1]。β折叠与α螺旋排列在一起以稳定整个折叠。高溶解度和热稳定性[2，3]使泛素成为通过物理化学分析研究蛋白质结构和动力学的标准分子（在参考文献1的介绍中总结）。[4]）。在生物学上，泛素通过与底物蛋白共价连接在控制各种细胞过程中起关键作用[5，6]。在许多情况下，一个泛素分子与另一个泛素分子连接以产生多聚泛素链来发挥其功能。一个突出的例子是蛋白酶体的蛋白质降解，其中通过Lys 48连接的多聚泛素链作为降解信号[5]。（聚）泛素化的酶级联反应包括一系列酶：泛素被泛素活化酶E1活化。然后，泛素缀合酶E2接收活化的泛素。最后，泛素连接酶E3帮助泛素转移到底物蛋白。许多泛素受体有助于将底物蛋白募集到蛋白酶体。在蛋白质水解发生之前，多聚遍在蛋白链通过蛋白酶体相关的去遍在蛋白酶与底物蛋白分离，然后作为游离遍在蛋白分子有效地回收。因此，泛素的自由和结合状态之间的平衡应该与其在细胞中的功能紧密结合。如果在核心位置的某些突变可以改变这种平衡而不对整体结构和热稳定性产生任何影响，则此类突变可能通过改变动力学来影响泛素功能。Bolon等人利用核磁共振（NMR）和圆二色性（CD）等光谱技术研究了泛素核心结构与动力学和功能的关系以及他们开发的用于研究泛素突变对酵母细胞的影响的体内生长测定[7]。先前开发的SUB 328酵母菌株中的泛素表达严格依赖于半乳糖。因此，用右旋糖培养基替换快速降低泛素表达水平并停止酵母生长，这可以通过引入组成型表达泛素的质粒来恢复。通过该拯救实验评估泛素的所有15个核心残基。在15个泛素突变体中，13个可以完全或部分支持酵母生长，而I30 A和L43 A突变体则不能。通过CD光谱法研究了这些突变体和其他生长支持突变体的热力学。从CD光谱估计的它们的解链温度表明I30 A和L43 A突变体在酵母细胞中良好折叠，类似于野生型和其他growthsupporting突变体。
Mutations at solvent-inaccessible core positions may impact protein functions by changing protein dynamics. However, such mutations may also change the global structure and/or thermodynamic stability of proteins, and therefore, their impacts are difficult to be interpreted in many cases. In this issue of the Journal of Molecular Biology, Fushman, Bolon and their colleagues challenged this difficulty studying ubiquitin as the reference molecule. Ubiquitin is a highly conserved 76-residue protein from yeast to mammals. The fold of ubiquitin consists of a five-stranded β-sheet, a short 310 helix and an α helix [1]. The β-sheet is lined with the α helix to stabilize the overall fold. High solubility and thermostability [2, 3] make ubiquitin a standard molecule for investigating protein structure and dynamics by physicochemical analyses (summarized in the introduction of Ref.[4]). Biologically, ubiquitin plays critical roles in control of various cellular processes through covalent attachment to substrate proteins [5, 6]. In many contexts, one ubiquitin molecule is linked to the other ubiquitin molecule to produce polyubiquitin chains to exert their functions. A prominent example is protein degradation by the proteasome, where polyubiquitin chains linked via Lys48 serve as the degradation signal [5]. The enzymatic cascade of (poly) ubiquitination includes a series of enzymes: Ubiquitin is activated by a ubiquitin-activating enzyme E1. Then, a ubiquitinconjugating enzyme E2 receives the activated ubiquitin. Finally, a ubiquitin ligase E3 aids transfer of the ubiquitin to substrate proteins. Many ubiquitin receptors contribute to recruitment of substrate proteins to the proteasome. Polyubiquitin chains are detached from substrate proteins by proteasomeassociated deubiquitinating enzymes before proteolysis occurs and then efficiently recycled as free ubiquitin molecules. Therefore, a balance between free and conjugated states of ubiquitin should be tightly coupled with its function in cells. If certain mutations at core positions could change this balance without any impact on the overall structure and thermostability, such mutations might affect ubiquitin function by altering the dynamics.Fushman, Bolon and their colleagues investigated relationship of ubiquitin core structures with dynamics and function in ubiquitin by using spectroscopic techniques of nuclear magnetic resonance (NMR) and circular dichroism (CD) and in vivo growth assay that they developed to study impacts of ubiquitin mutations on yeast cells [7]. Ubiquitin expression in the previously developed SUB328 yeast strain strictly depends on galactose. Therefore, replacement with dextrose media rapidly decreases ubiquitin expression level and stops the yeast growth, which can be recovered by introducing a plasmid that constitutively express ubiquitin. All 15 core residues of ubiquitin were assessed by this rescue experiment. Of 15 ubiquitin mutants, 13 could fully or partly support yeast growth, whereas I30A and L43A mutants failed. Thermodynamics of these mutants and other growth-supporting mutants was investigated by CD spectrometry. Their melting temperatures estimated from the CD spectra indicate that I30A and L43A mutants are well folded in yeast cells, similarly to wild type and other growthsupporting mutants.