冷凍電子顯微術用於蚯蚓血紅蛋白結構之研究

Abstract

蚯蚓血紅蛋白是屬於胞外型的氧氣運輸蛋白，對於氧氣的結合具有非常高的協同效應，科學家相信它們的攜氧機制有別於脊椎動物和其它無脊椎動物。然而，由於缺乏結構上的分析去探討蚯蚓血紅蛋白攜帶不同氣體配體時的結構差異，我們對於它們的協同結合機制所知甚少。在此篇論文中，利用冷凍電子顯微鏡的技術，解析出普通蚯蚓 (Lumbricus terrestris) 血紅蛋白在生理環境下結合氧氣時的結構，此結構之解析度有 8.1 埃的分辨率，利用分子動態擬合程序，建立了一個準原子模型，此攜氧的模型和已知攜帶一氧化碳的 X-ray 結構有顯著的差異。藉由比較分析不同氣體配體時的結構，結果首度發現了要能充分解釋其協同結合機制，蚯蚓血紅蛋白在血基質基團附近有三級和四級結構的改變，另外也有整體的外擴產生，此整體的結構變化是藉由內環和跨環接觸之輔助所達成。相較於脊椎動物和其它無脊椎動物，蚯蚓血紅蛋白在協同效應的功能上有更大的複雜性。此外，由冷凍電鏡結構清楚地解析了額外的中央手鐲結構，它對於穩固整個複合體的結構扮演很重要的角色，並且對於長久以來 X-ray 結晶結構所缺失的中央電子雲密度之爭論，提供更加明確的解釋。 利用蚯蚓血紅蛋白的輸血醫學研究已經在動物實驗進行中，科學家發現在極度貧血的狀況，蚯蚓血紅蛋白能增加氧氣的承載能力，且不會引發嚴重的副作用，在本研究中，針對蚯蚓血紅蛋白協同攜氧與中央手鐲結構輔助組裝的機制提供了更清楚的結構證據，這對於科學家利用此蛋白研發醫療試劑有很大的助益，從此研究工作中獲得的結構資訊將會是在醫療與製藥相關進一步的體內和體外實驗一個重要的里程碑。

The earthworm hemoglobins (Hbs) are extracellular oxygen-carrying proteins with unusually high cooperativity of ligand binding. It was believed that their mechanism of oxygen-binding is quite different from the vertebrate and other invertebrate Hbs. However, the cooperative binding mechanisms are still mostly unknown due to the lack of the structural analysis between different ligand states. In this dissertation, the cryo-electron microscopy (cryo-EM) structure of the common earthworm (Lumbricus terrestris) Hb in its native, oxygenated state at 8.1 Å resolution was reported. A pseudo-atomic model was built by flexible fitting procedures showing remarkable differences from the CO-binding structure. The structures in the different functional states first indicated that to fully express cooperative ligand binding, the L. terrestris Hb required unique tertiary and quaternary transitions in the heme pocket and a global subunit movement facilitated by intra-ring and inter-ring contacts. The results revealed greater complexity in cooperative function of L. terrestris Hb than the vertebrate and other invertebrate Hbs. Moreover, the cryo-EM structure clearly revealed the existence of additional sinusoidal bracelet which played an important role in stabilizing the central linker complex and provided the confirmation for the long-standing debate about the additional electron densities absent in the X-ray crystal structure. Transfusion studies in animals are in progress and have shown increased O2 carrying capacity during extreme anemia without causing severe side effect. In this study, results provided better understanding of the molecular mechanism of cooperative O2 binding and bracelet-assisted assembly of earthworm Hb. This paves the way for scientists to develop earthworm Hb-based reagents for medical treatments. The structural information gained from this work is a milestone for further in vitro and in vivo studies in medical and pharmaceutical applications.