第一部分：台灣眼鏡蛇毒金屬蛋白水解酵素與αvβ3整合素結合模式之研究
我們實驗室先前從台灣眼鏡蛇毒中純化出兩種個別採取不同結構型態(C and I 型態)的蛇毒金屬蛋白水解酵素，分別是atragin 和Kaouthiagin-like。這兩種蛇毒金屬蛋白水解酵素的結構及序列都包含了金屬水解酵素區域、去整合素相似區域、多半胱氨酸區域，是屬於PIII 類型的蛇毒金屬蛋白水解酵素。在此，我們將探討此兩種台灣眼鏡蛇蛇毒金屬蛋白水解酵素在抑制傷口癒合的過程中扮演的角色。我們發現C 型態不含RGD氨基酸序列的atragin與αvβ3 整合素的結合親和力是比I 型態且含RGD氨基酸序列的Kaouthiagin-like與αvβ3整合素的親和力的十倍。而atragin是採取一種新穎的結合模式和整合素結合，這結合模式主要是透過atragin的金屬水解酵素區域以及在半胱氨酸區域上的RRN氨基酸序列。在細胞貼附實驗中，顯示纖維母細胞主要是透過細胞上的αvβ3整合素與atragin結合貼附。再更進一步的傷口癒合實驗中發現atragin可以透過和αvβ3整合素結合去抑制傷口癒合及細胞移動。這些結果顯示除了實驗室之前發現的CTX A5之外，在台灣眼鏡蛇中還有許多不含RGD氨基酸序列的蛋白質可以特定地與整合素結合而更進一步去抑制傷者的傷口癒合過程。

第二部分： LonA蛋白水解酵素晶體結構分析
LonA 蛋白水解酵素是一種演化上保留下的的酵素存在於真核及原核生物，主要是利用水解ATP的活性去造成結構上改變進一步將其受質移動及降解。而LonA的受質主要是細胞內一些受到破壞而造成結構錯誤的蛋白質還有些特定的調控蛋白。LonA蛋白水解酵素擁有一個特色就是其受質可以刺激活化LonA蛋白水解酵素本身的水解ATP的活性。在此，我們發表了一個六聚體的LonA蛋白水解酵素的結構，此結構中存在著ADP的單體及不含ADP的單體，彼此間隔依序排列成六聚體。核酸的結合造成彼此不相鄰的單體上的poor loops、蛋白水解groove以及之前所不知的Arg-paddle的結構上的巨大移動。結構及生化實驗的結果顯示，受質結合的蛋白水解區域的groove可以異位性刺激LonA的水解ATP活性，以及Arg-paddle可以促進蛋白質受質降解。這些實驗數據提供了一個我們可以去了解LonA如何利用ATP水解造成結構上改變進而去異位性影響其受質的降解。

Part. I: Binding model analysis of snake venom metalloproteinase/αvβ3 integrin complex

We have previously identified two new P-III type ADAM-like snake venom metalloproteinases (SVMPs), i.e., atragin and kaouthiagin-like, from Taiwan cobra venom and determined their 3D structures with a distinct C- and I-shaped metalloproteinase/disintegrin-like/cysteine-rich (MDC) modular architecture. Herein, we investigated their functional targets to elucidate the role of cobra SVMPs in perturbing wound healing in snakebite victims. We showed that the non-RGD (Arg-Gly-Asp) C-shaped SVMP atragin binds about ten-fold stronger than the RGD-containing I-shaped SVMP kaouthiagin-like to αvβ3 integrin in the surface-immobilized form. Atragin binds to αvβ3 integrin through a novel interaction mode involving distal M and C domains via the RRN sequence motif in the hyper variable loop. In a cell adhesion assay, the adhesion of fibroblasts to atragin was mediated by αvβ3 integrin. Furthermore, atragin inhibited wound healing and suppressed cell migration in an αvβ3 integrin-dependent manner. These results, together with our previous demonstration of non-cytotoxic cobra CTX A5 in targeting αvβ3 integrin, suggest that cobra venom consists of several non-RGD toxins with integrin-binding specificity that could perturb wound healing in snakebite victims.

Part. II: The crystal structure of the Lon AAA+ protease

The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg-paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg-paddle in driving substrate degradation. Together, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine.

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