在最新一期的《自然》雜志上，來自華盛頓大學(xué)的華裔科研人員鄭寧（Ning Zheng）助理教授又發(fā)表了一篇有關(guān)泛素蛋白連接酶結(jié)構(gòu)生物學(xué)的新文章,。自2000年以來,，鄭博士先后在Cell、Nature和Science等國際權(quán)威雜志上發(fā)表了多篇文章,，并且有三篇文章成為雜志的封面故事進(jìn)行推薦。
蛋白質(zhì)泛素化作用是后翻譯修飾的一種常見形式，該過程能夠調(diào)節(jié)不同細(xì)胞途徑中各式各樣的蛋白質(zhì)底物,。通過一個三酶級聯(lián)（E1-E2-E3），蛋白質(zhì)的泛酸連接又E3泛素連接酶催化,，這種酶是cullin-RING復(fù)合體超級家族的最佳代表,。
在從酵母到人類的各級生物中都保守的DDB1-CUL4-ROC1復(fù)合體是最近確定出的cullin-RING泛素連接酶，這種酶調(diào)節(jié)DNA的修復(fù),、DNA復(fù)制和轉(zhuǎn)錄,，它能被病毒所破壞。
由于缺少一個規(guī)則的SKP1類cullin連接器和一種確定的底物召集結(jié)構(gòu)域,，目前人們還不清楚DDB1-CUL4-ROC1 E3復(fù)合體如何被裝配起來以對各種蛋白質(zhì)底物進(jìn)行泛素化,。
在這項(xiàng)新的研究中,，鄭博士等人對人類DDB1-CUL4A-ROC1復(fù)合體被病毒劫持的形式進(jìn)行了晶體結(jié)構(gòu)分析。分析結(jié)果表明DDB1利用一個β-propeller結(jié)構(gòu)域作為cullin骨架結(jié)合物,，利用一種多變的,、附著的獨(dú)立雙β-propeller折疊來進(jìn)行底物的呈遞。
通過對人類的DDB1和CUL4A復(fù)合體進(jìn)行聯(lián)系提純,，然后進(jìn)行質(zhì)譜分析,，研究人員確定出了一種新穎的WD40-repeat蛋白家族，這類蛋白直接與DDB1的雙propeller折疊結(jié)合并充當(dāng)E3酶的底物募集模塊,。這些結(jié)構(gòu)和蛋白質(zhì)組學(xué)研究結(jié)果揭示出了cullin-RING E3復(fù)合體的一個新家族的裝配和多功能型背后的結(jié)構(gòu)機(jī)制和分子邏輯關(guān)系,。
Dr. Ning Zheng
Assistant Professor
Structural Biology of Ubiquitin-Protein Ligases
Protein turnover represents an efficient mechanism in regulation of protein functions in almost all areas of biology. Protein ubiquitination and ubiquitination-dependent proteolysis play a central role in controlling protein turnover. Their importance in biology is being recognized based on recently fast growing studies in areas such as cell cycle control, DNA repair, transcription, signal transduction, and apoptosis. Deregulation of ubiquitination pathways often leads to abnormal cell growth and differentiation and is linked to cancer and a number of diseases. The biological significance of protein ubiquitination is almost comparable to that of protein phosphorylation.
To degrade a protein in a specific and timely manner, eukaryotic cells first covalently modify the protein substrates on their lysine residues with a small highly conserved protein, ubiquitin (Figure 1). Additional ubiquitin molecules are then added in a sucessive way, forming a polyubiquitin chain. Polyubiquitinated protein substrates are targeted to the proteasome, which mediates the proteolytic digestion of the substrates. Three classes of enzymes are involved in the ubiquitination reactions. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3). The specificity and extension of ubiquitin transfer from E2 to the substrate is mediated by the E3, which on the one hand recognizes a specific substrate and on the other hand recruits an charged E2.
Figure 1. The E1-E2-E3 cascade in the ubiquitination pathway.
In the past several years, a large family of ubiquitin-protein ligases, the RING E3s, has been discovered. The RING E3s all share a RING-type zinc finger motif, either in the same polypeptide which contains substrate binding domain, or as a subunit in a multi-subunit protein complex. Single polypeptide RING E3s with demonstrated E3 activities include a number of key regulatory proteins in human cells, such as c-Cbl (E3 for tyrosine kinases), mdm2 (E3 for p53), BRCA1 (breast cancer associated gene) , parkin (Parkinson disease gene), and IAPs (Inhibitor of apoptosis protein). A few multi-subunit E3 complexes have also been identified. Among them, the SCF complexes represents a super subfamily of RING E3s, which mediates ubiquitination of a wide range of protein substrates such as p27, IkappaB, and beta-Catenin.
We have been interested in understanding the structure and function of the ubiquitin-protein E3 ligases. Using X-ray crystallography combined with biochemical analyses, we have characterized and determined the structure of c-Cbl E3 in complex with an E2 (Figure 2) and a quaternary SCF-Skp2 E3 complex (Figure 3). These structures not only show how the RING finger recruits a ubiquitin-conjugating enzyme E2 but also reveal its spatial relationship with the rest of the E3, providing the structural basis of how RING type ubiquitin protein ligases mediate the ubiquitin transfer reaction. Our future studies will focus on understanding at the structural level how the RING E3s are regulated and how they play a role in biological functions such as DNA repair, transcription, and protein quality control. The long term goal of Zheng's lab is to derive and use structural information of protein complexes to predict and manipulate protein functions. For example, we are currently developing new techniques to search for the unknown substrates of many potential RING E3s based on the structural information we obtained with c-Cbl and SCF.
Figure 2. Superposition of c-Cbl-E2 and E6AP-E2 complex structures. The two types of E3s (RING and HECT) recognize the same structural elements of the E2 in spite of their distinct sequence and structure motives.
Figure 3. A model of SCF-Skp2-E2 complex based on the crystal structure of the quaternary complex of SCF-Skp2Fbox. Skp2's Leucine-Rich-Repeat domain binds its protein substrate and presents it to the E2's active site, where the ubiquitin is anchored through a thioester bond.
Publications
T. Li, X. Chen, K.C. Garbutt, P. Zhou, and Ning Zheng 2006. Crystal structure of DDB1 in complex with simian virus 5 V protein: A multifacet propeller cluster in ubiquitin ligase machinery under viral hijack. Cell 124:105-117.
Zheng, N., Schulman, B.A., Miller, J.J., Wang, P., Jeffrey, P.D., Chu, C., Koepp, D.M., Elledge, S.J., Pagano, M., Conaway, R.C., Conaway, J.W., Harper, J.W., and Pavletich, N.P. (2002) Structure of the Cul1-Rbx1-Skp1-FboxSkp2 Ubiquitin-Protein Ligase Complex. Nature 416: 703-709 (Cover Story).
Yang, H., Jeffrey, P.D., Miller, J.J., Kinnucan, E.R., Sun, Y., Tomas, N.H., Zheng, N., Chen, P., Lee, W.H., and Pavletich, N.P. (2002) BRCA2 Function in DNA Binding and Recombination from a BRCA2-DSS1-ssDNA Structure. Science 297: 1837-1848 (Cover Story).
Zheng, N., Wang, P., Jeffrey, P.D., and Pavletich, N.P. (2000) Structure of a c-Cbl-UbcH7 Complex: RING Domain Function in Ubiquitin-Protein Ligases. Cell 102: 533-539.
Zheng, N., Fraenkel, E., Pabo, C.O., and Pavletich, N.P. (1999) Structural Basis of DNA Recognition by the Heterdimeric Cell Cycle Transcription Factor E2F-DP. Genes & Development 13: 666-674 (Cover Story).
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