核糖核酸水解酶PNPase突變導致疾病之結構解析

Structural insights into the disease-linked human PNPase mutants in RNA binding and degradation

郭巴耳(Golzarroshan, Bagher)

指導教授 ： 袁小琀 ； 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2018年)

摘要

Polynucleotide phosphorylase (PNPase) 是一演化上具高度保守性的核糖核酸外切酶，主要參與原核生物與真核生物中的RNA加工與降解作用。人類PNPase主要存在於粒線體中，除了參與RNA加工與降解作用之外，同時還參與引導具有結構的核糖核酸，進入粒線體基質 (mitochondria matrix) 中，包含5S rRNA, MRP RNA, RNase P RNA。PNPase的基因突變導致許多與人類粒線體功能缺失相關的疾病，突變造成粒線體內RNA分子的累積，或者破壞PNPase引導核糖核酸進入粒線體基質的功能。結晶學研究的結果已知PNPase為一三聚體環狀結構的蛋白，中間會形成一個單股RNA結合的通道，RNA的3端端點會由此通道被引導至PNPase的活性位置進行降解。然而目前對於疾病相關的基因突變，對PNPase蛋白的三級結構、四級結構的組成以及活性的影響仍並不清楚。
在本研究當中，我們發現一些疾病相關的胺基酸點突變，例如Q378R與E475G，會破壞PNPase的三聚體結構。具有這些突變的PNPase會形成二聚體結構，且對RNA結合與降解的活性較正常的PNPase活性低。此外，我們發現PNPase的S1結構區域主要負責結合具有結構的核糖核酸RNA的莖環 (stem-loop) 區域，而非RNA單股區域。我們更進一步解析了PNPase二聚體的晶體結構，結構的解析度為2.8 Å，其中的PNPase單體去除了S1結構區域並帶有N段組胺酸標籤 (His-Tag)。PNPase的結構顯示KH的活性區域，在二聚體的結構中比三聚體的結構更加封閉。我們也將二聚體以及三聚體的PNPase透過X光小角度散射 (Small-angle X-ray Scattering, SAXS) 實驗進行測定。結果顯示，S1活性區域在兩種結構當中位置變動性很大，可以看到開放與封閉的兩種構型。而在二聚體PNPase當中，S1活性域也較為封閉。實驗結果顯示，二聚體PNPase由於參與RNA結合的S1與KH的活性區域較為封閉，因此造成其與RNA的結合能力降低。總結上述的實驗結果，我們發現若胺基酸的點突變位於PNPase形成三聚體的結構中，單體與單體之間的交界處，則會造成PNPase趨向於形成二聚體的結構，並且形成較封閉的RNA結合通道。如此會導致PNPase對於RNA引導與降解的活性下降，進而造成粒線體功能異常與疾病。本研究也提供了治療與PNPase功能相關疾病的資訊以及可能的方法，例如藉由穩定PNPase三聚體的結構促進其RNA引導與降解的活性，而達到治療疾病的效果。

關鍵字

RNA 降解 ； 粒線體RNA 導入 ； 粒線體蛋白 ； 核醣核酸酶 ； 蛋白質結構

並列摘要

Polynucleotide phosphorylase (PNPase) is an evolutionary conserved 3'-to-5' exoribonuclease that functions in RNA processing and turnover in prokaryotes and eukaryotes. Human PNPase is mainly located in mitochondria where it not only participates in RNA processing and decay but it is also involved in importing a subgroup of structured RNAs, including 5S rRNA, MRP RNA, RNase P RNA and miRNAs, into the mitochondrial matrix. Mutations in PNPase that impair either mitochondrial RNA (mtRNA) degradation or RNA import are thus connected to mitochondrial dysfunctions and a wide spectrum of human diseases. Crystal structural studies reveal that PNPase is a trimeric protein assembled into a ring-like structure with a central channel for binding of a single-stranded RNA (ssRNA) and guiding its 3' end into the active site for degradation. However, it remained unclear how the disease-linked PNPase mutations affect protein folding, assembly and/or enzymatic activity.
In this study, we show that the trimeric assembly of PNPase is disrupted by the disease-linked mutations, including Q378R and E475G. PNPase is oligomerized into a dimeric conformation after introducing the disease-linked mutations, and these PNPase mutants exhibit lower RNA-binding and degrading activities as compared to the wild-type protein. Moreover, we found that S1 domain of PNPase is responsible for the interaction with the stem-loop motif of imported RNAs but it is not involved in binding to ssRNA. We further determined the crystal structure of the dimeric form of the S1 domain-truncated PNPase with an N-terminal His-tag at a resolution of 2.8 Å showing that the KH domains are less accessible in the dimeric form of PNPase. The overall structures of the full-length trimeric and dimeric forms of PNPase were further determined by small-angle X-ray scattering (SAXS), showing that S1 domains are flexible with open to closed conformations in both conformers and that S1 domains are not fully accessible in the dimeric structure. Taken together these results explain why these dimeric PNPase mutants with less accessible RNA-binding KH and S1 domains interact with RNA poorly. We thus conclude that mutations at the interface of the trimeric PNPase tend to produce a dimeric protein with obstructed RNA-binding surfaces, thus impairing both of its RNA import and degradation activities and leading to mitochondria dysfunction and diseases. This study provides a possible strategy for the treatment of PNPase-associated diseases by stabilizing the trimeric conformation of PNPase that could improve its functions in both RNA import and decay in mitochondria.

並列關鍵字

RNA decay ； mitochondrial RNA import ； mitochondrial proteins ； exoribonuclease ； crystal structure

參考文獻

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