Mitrea Diana M, Kriwacki Richard W
St. Jude Children's Research Hospital, Department of Structural Biology, Memphis, TN 38103, USA.
Pac Symp Biocomput. 2012:152-63.
It is now well appreciated that disordered proteins and domains are prevalent in eukaryotic proteomes and that disorder is critically linked with their regulation and functionality. However, our recent observations with the multi-domain protein, nucleophosmin (Npm), suggest that the biological palette of disorder is more diverse than currently understood. The N-terminal oligomerization domain of Npm (Npm-N) can be transformed from a folded, pentameric structure to a monomeric, disordered state through changes in solution ionic strength and, importantly, through physiologically relevant post-translational modifications. Thus, it appears that Npm has been evolutionarily tuned to exist in equilibrium between disordered and ordered states. Results from us and others suggest that the function of Npm is regulated through shifts in this equilibrium via post-translational modifications. Interestingly, this polymorphic behavior is not detected using standard secondary structure and disorder prediction algorithms, which show Npm-N to be folded into β-strands, consistent with the structure of the pentameric form. We have used a combination of computational tools, including structure-based analysis, sequence analysis algorithms (NetPhos 1.0, SCRATCH, KinasePhos, GPS2.1, PONDR) and molecular mechanics energy calculations, to test the hypothesis that the polymorphic behavior of Npm-N can be understood on structural and energetic grounds. This computational strategy has resulted in the identification of unfavorable energetic "hot-spots" within the Npm-N structure which coincide with experimentally observed sites of post-translational modification. Based on these observations, we propose that Npm-N has evolved energetic switches within its structure to enable transformation to a disordered state through phosphorylation. We further propose that the transformation process is triggered by sequential phosphorylation of solvent exposed hot-spots followed by exposure and modification of additional but initially buried sites to completely shift the equilibrium to the disordered state. This regulated, shifting equilibrium is associated with control of Npm localization within the nucleolus, nucleoplasm and cytoplasm, and with its role in regulation of centrosome duplication through interactions with Crm1-Ran. More broadly, we present a general computational strategy to identify transformational hot-spots within proteins and to test the hypothesis that other proteins currently understood to be folded participate in functionally-relevant order-disorder equilibria as we have observed for Npm. The identification of such polymorphic proteins would broaden the palette of protein disorder utilized in biological systems.
如今人们已经充分认识到，无序蛋白质和结构域在真核生物蛋白质组中普遍存在，而且无序状态与其调控及功能密切相关。然而，我们最近对多结构域蛋白核磷蛋白（Npm）的观察表明，无序状态的生物学表现形式比目前所理解的更加多样。Npm的N端寡聚化结构域（Npm-N）可以通过溶液离子强度的变化，更重要的是通过生理相关的翻译后修饰，从折叠的五聚体结构转变为单体无序状态。因此，似乎Npm在进化过程中已被调整为在无序和有序状态之间保持平衡。我们和其他人的研究结果表明，Npm的功能是通过翻译后修饰改变这种平衡来调节的。有趣的是，使用标准的二级结构和无序预测算法无法检测到这种多态行为，这些算法显示Npm-N折叠成β链，与五聚体形式的结构一致。我们使用了多种计算工具，包括基于结构的分析、序列分析算法（NetPhos 1.0、SCRATCH、KinasePhos、GPS2.1、PONDR）和分子力学能量计算，来检验Npm-N的多态行为可以从结构和能量角度来理解这一假设。这种计算策略已经识别出Npm-N结构内不利的能量“热点”，这些热点与实验观察到的翻译后修饰位点一致。基于这些观察结果，我们提出Npm-N在其结构中进化出了能量开关，以使其能够通过磷酸化转变为无序状态。我们进一步提出，转变过程是由溶剂暴露的热点依次磷酸化，随后是其他最初埋藏位点的暴露和修饰，从而将平衡完全转移到无序状态所触发的。这种受调控的、不断变化的平衡与Npm在核仁、核质和细胞质中的定位控制有关，也与其通过与Crm1-Ran相互作用在中心体复制调控中的作用有关。更广泛地说，我们提出了一种通用的计算策略，用于识别蛋白质中的转变热点，并检验其他目前被认为是折叠状态的蛋白质是否像我们观察到的Npm那样参与功能相关的有序-无序平衡这一假设。识别出这类多态蛋白质将拓宽生物系统中利用的蛋白质无序状态的范围。
