Nature：郑宁破译泛素蛋白连接酶复合物的结构
蛋白质泛素化作用是后翻译修饰的一种常见形式，该过程能够调节不同细胞途径中各式各样的蛋白质底物。通过一个三酶级联（E1-E2-E3），蛋白质的泛酸连接又E3泛素连接酶催化，这种酶是cullin-RING复合体超级家族的最佳代表。
在从酵母到人类的各级生物中都保守的DDB1-CUL4-ROC1复合体是最近确定出的cullin-RING泛素连接酶，这种酶调节DNA的修复、DNA复制和转录，它能被病毒所破坏。
由于缺少一个规则的SKP1类cullin连接器和一种确定的底物召集结构域，目前人们还不清楚DDB1-CUL4-ROC1 E3复合体如何被装配起来以对各种蛋白质底物进行泛素化。
在这项新的研究中，郑博士等人对人类DDB1-CUL4A-ROC1复合体被病毒劫持的形式进行了晶体结构分析。分析结果表明DDB1利用一个β-propeller结构域作为cullin骨架结合物，利用一种多变的、附着的独立双β-propeller折叠来进行底物的呈递。
通过对人类的DDB1和CUL4A复合体进行联系提纯，然后进行质谱分析，研究人员确定出了一种新颖的WD40-repeat蛋白家族，这类蛋白直接与DDB1的双propeller折叠结合并充当E3酶的底物募集模块。这些结构和蛋白质组学研究结果揭示出了cullin-RING E3复合体的一个新家族的装配和多功能型背后的结构机制和分子逻辑关系。
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).
E-421 Health Sciences Building
Box 357280
Seattle WA 98195-7280
Phone: (206) 616-3990; Fax: (206) 685-3822
nzheng@u.washington.edu
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