行政院國家科學委員會專題研究計畫 期中進度報告
間白質增因子與 DNA 修復蛋白 HHR23A 的結構與功能之研究
(2/3)
計畫類別： 個別型計畫
計畫編號： NSC92-2311-B-006-006-
執行期間： 92 年 08 月 01 日至 93 年 07 月 31 日 執行單位： 國立成功大學生物化學科（所）
計畫主持人： 莊偉哲
報告類型： 精簡報告
處理方式： 本計畫涉及專利或其他智慧財產權，1 年後可公開查詢
中 華 民 國 93 年 5 月 17 日
行政院國家科學委員會補助專題研究計畫成果報告
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間白質增因子與 DNA 修復蛋白 HHR23A 的結構與功能之研究 (2/3)
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計畫類別： 整合型計畫
計畫編號： NSC 92-2311-B-006-006
執行期間： 92 年 8 月 1 日至 93 年 7 月 31 日
計畫主持人：莊偉哲
共同主持人：
計畫參與人員：
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
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□國際合作研究計畫國外研究報告書一份
執行單位：成大醫學院生化所
中 華 民 國 93 年 5 月 31 日
行政院國家科學委員會專題研究計畫成果報告
行政院國家科學委員會專題研究計畫成果報告
題目:
間白質增因子與 DNA 修復蛋白 HHR23A 的結構與功能之研究 (2/3)計畫編號：NSC 92-2311-B-006-006
執行期限：92 年 8 月 1 日至 93 年 7 月 31 日
主持人：莊偉哲 成大醫學院生化所
一、中文摘要
間 白 素 結 合 因 子 (Interleukin enhancer binding factor, ILF) 是一個從 Hela 與 Jurkat cDNA 基因庫中所選殖出 來的細胞轉錄因子，全長含有 655 個胺基 酸，分子量為 72 kDa。在功能上，ILF 藉 著其 DNA 結合區與人類後天免疫不全病 毒 I 型長端點重複部分(HIV-1 LTR)及間白 素 II 型 啟 動 子 (IL-2 promoter) 的 purine-rich motif 結合，進而活化 HIV-1 與 IL-2 基因表現。ILF 的 DNA 結合區經由 序 列 比 對 與 winged helix/forkhead (WH/FOX) 轉 錄 因 子 有 35~89% 的 相似 性，因而將 ILF 歸類為 WH/FOX 家族之蛋 白。我們藉二級結構預測發現，由 ILF 的 DNA 結合區往 N 端延伸，找到可能形成 2 個α-helix 結構的區域。因此推測 ILF 也許 具有類似 Ets-1 的自我抑制機制，亦即利 用 C 端 Helix4 與 N 端延伸部分的 2 個 α-helix 形成所謂 inhibitory module，來調 節與 DNA 結合。所以我們構築出不同長度 片段的 ILF，經質譜鑑定確認純化到正確的 蛋白質。
在 功 能 方 面 ， 我 們 利 用 EMSA (electrophoretic mobility shift assay)，研 究不同片段 ILF 對不同 DNA 的結合能力。
發現 ILF 與含有 TGTTTAC 或 TGTTTTG 序列的 DNA 有較強的結合力。進一步，我 們以四株 ILF 片段與 WRT 結合，結果發 現我們構築的 ILF 片段對 WRT 的結合 能力如下： ILF111(98) > ILF117(104) >
ILF111(92∆6) ~ ILF105(92) 。因前兩株皆 含有 NLS，後兩株無 NLS。由此結果，
我們推測 ILF DNA 結合區域需含 98 個 胺基酸且必須包含 NLS，而 C 端區域片 段可能會抑制 ILF 與 DNA 之結合。 在
結構方面，我們利用 NMR 完成 ILF 的 DNA 結合區於水溶液中之 3D 結構分析方 面，目前我們正進行 ILF 與 DNA 複合體的 3D 結構的分析。
DNA 修復蛋白 HHR23A 和 HHR23B 是人類與酵毋菌 Rad23 的同糸蛋白。到目 前為止我們對 HHR23A 與 HHR23B 的功 能不是很明瞭。而酵毋菌中的 Rad23 幫助 核苷酸切除修復蛋白質複合體之組合，因 此在核苷酸切除修復機制扮演重要的角 色。HHR23A 和 HHR23B 可與 DNA 修復 酵素缺乏蛋白(XPC)作用，幫助 XPC 在核 苷酸修復的活性。HHR23A 與 HHR23B 之 間有 60%同一性，分別含有 363 與 408 個 殘基。包括 HHR23A 與 HHR23B 酵毋菌 Rad23 的同糸蛋白具有相同的功能部位結 構，包含一個泛素(ubiquitin)區，二個泛素 結合區，一個 XPC 區。我們表現 HHR23A 的 UBL， UBA1，UBA2 三個功能部位並 確定這三個蛋白摺疊正確。目前我們正進 行 HHR23A 三個功能部位的 3D 結構的分 析。
關鍵詞：動力，結構，核磁共振，間白素 結合因子，DNA 修復蛋白 HHR23A。
Abstract
Interleukin binding factor (ILF) is a cellular transcription factor isolating from Hela and Jurkat cDNA library. ILF binds to purine-rich regulatory motifs in both HIV-1 LTR and the interleukin-2 promoter. So far, two ILFs were found.
They are designated as ILF-1 and ILF-2 and have 655 and 609 amino acids, respectively. Both ILF-1 and ILF-2 contain several amino acid homologies, including a region for potential
nucleotide-binding site, a nuclear localization signal, an N-glycosylation motif, and a DNA-binding domain.
Sequence analysis reveals that ILF contains several homologous regions including a nucleotide-binding site, a DNA-binding domain, a potential nuclear localization signal (NLS), an ubiquitin-mediated degradation site, and an N-glycosylation site. The DNA-binding domain of ILF has more than 60%
similarity with that of Drosophila fork head domain and hepatocyte-specific transcription factor-3 (HNF-3), and 89%
identity with that of Myocyte nuclear factor (MNF).
The DNA repair proteins HHR23A and HHR23B are the human homologues of the yeast Rad23 protein that functions in nucleotide excision repair. However, little is known about the biochemical function of HHR23A and HHR23B. In yeast, Rad23 promotes the assembly of a multiple protein complex involved in nucleotide excision. Both HHR23A and HHR23B interact with the Xeroderma pigmentosum group C (XPC) protein and promote the DNA repair activity of XPC. HHR23A and HHR23B share 60% identity and contain 363 and 408 residues, respectively. All of the Rad23 homologues including HHR23A and HHR23B share common domain structures, including one ubiquitin-like domain (UBL), two ubiquitin-associated domains (UBA1 and UBA2), and one XPC domain.
In order to study the structure and function relationships of ILF and HHR23A, we expressed four various sizes of ILF including ILF105(92), ILF111(98), ILF111(92∆6) and ILF117 (104), as well as the UBA1, UBA2, and UBL domains of HHR23A. These recombinant proteins have been purified to be homogeneous. In the functional studies ， we used gel retardation analysis to study the interaction between various lengths of DNA-binding region of ILF and motifs of DNA. The retardation
study showed that the relative binding affinities of the four proteins to the DNA probe were ILF111(98) > ILF117(104) >
ILF105(92) and ILF111(92∆6)
In the structural studies， we used NMR spectroscopy to determine the 3D structure of the DNA-binding domain of ILF. Determinations of 3D structures of the complex of the DNA-binding domain of ILF and DNA, as well as the UBA1, UBA2, and UBL domains of HHR23A are on going.
二、緣由與目的
The understandings of structure and function relationships of the interaction of proteins with DNA are essential in biological system. NMR spectroscopy is a powerful tool to investigate the three dimensional structures and dynamic properties of proteins; CD spectroscopy is useful to determine the secondary structures of proteins; and CD, and NMR spectroscopy and gel-retardation assay can be used to determine the binding between protein and DNA and the dissociation constant of them.
Combination of these spectroscopic methods, molecular cloning, and functional assay, we are able to understand the interaction between ILF and DNA in detail and to design the transcriptional drug based on the structural and functional studies.
The long-term objectives of this project are to study the structure and function relationships and dynamic properties of interleukin enhancer factor (ILF) and ILF/DNA complex. Since ILF can inhibit the replication of HIV-1, the resulting structures can be used to design the transcriptional drugs for targeting LTR of HIV-1. To date, more than 150 winged-helix proteins have been identified and ILF belongs to this family. They possess both a remarkable sequence homology in DNA-binding region and an equally notable variability in recognizing specific DNA. However,
little is known about their diverse DNA-binding properties with similar tertiary fold. In the study of ILF, we will (1) express various domains of ILF
(221-351, 211-351, and 202-354) and determine their domain structures and dynamics by NMR,
(2) combine gel retardation assay and CD titration to characterize what DNA sequence recognized by the DNA-binding domain of ILF,
(3) determine solution structures and dynamic properties of the ILF/DNA complex,
(4) phosphorylate various domain of ILF (221-351, 211-351, and 202-354) and determine the effect of phosphorylation on their structures, and
(5) determine the structure of ILF (202-354) in the presence of ATP.
In the study of HHR23A, we will
(1) express HHR23A (156-363) and determine its structures and dynamics by NMR,
(2) determine solution structure and dynamic property of UBA1, and
(3) determine solution structures and dynamic properties of the complexes of UBL/UBA1 and UBL/UBA2 of HHR23A.
三、結果與討論
1. The determination of 3D structure of the DNA-binding domain of
ILF/DNA (5’-TTGATGTTTATTGTT) complex by NMR spectroscopy is ongoing.
Figure 1. HSQC spectrum of ILF/DNA complex
2. The determination of 3D structure of the regulatory and DNA-binding domains of ILF by NMR spectroscopy is ongoing.
Figure 2. HSQC spectrum of ILF132
3. Various fragments of ILF have been expressed in E. coli and purified We have successfully expressed and purified six ILF fragments; ILF93(92) (residues 251-342); ILF99(98) (residues 251-348); ILF102(101) (residues
251-351); ILF132(131) (residues 221-351); ILF141 (residues 211-351);
and ILF153 (residues 202-354).
四、計畫成果自評
Combination of these spectroscopic methods, molecular cloning, and functional assay, we are able to understand the structure and function relationships of ILF and DNA. This study will expand our understanding of the molecular determinants of winged helix family involved in DNA binding and the role of HHR23A in DNA repair. In this proposal, the studies on winged
helix/forkhead proteins and HHR23A will make important contributions in the field of structural genomics in Taiwan.
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