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Volumn 286, Issue 5443, 1999, Pages 1321-1326

Structure of an E6AP-UbcH7 complex: Insights into ubiquitination by the E2-E3 enzyme cascade

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN P53; UBIQUITIN CONJUGATING ENZYME; UBIQUITIN PROTEIN LIGASE;

EID: 0032741446     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5443.1321     Document Type: Article
Times cited : (473)

References (51)
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    • 2- and COOH-termini, respectively. The model was built into the electron density map with O (40) and was refined against the 2.8 Å native data set with the program CNS (41) (Table 1).
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    • 1, with a = 103.4 Å, b = 112.7 Å, c = 123.8 Å, which is the same space group with closely related unit cell parameters as the hect domain-alone crystals. They contain the same trimeric form of the hect domain, and only one of the hect domains is bound by UbcH7. The UbcH7-binding site of a second hect domain is involved in crystal-packing contacts, and that of a third hect domain has little space for UbcH7 owing to crystal packing. This stoichiometry is consistent with that obtained by running extensively washed crystals in an SDS gel. The crystals of the complex were flash-frozen in crystallization buffer supplemented with 35% (v/v) ethylene glycol. The structure of the hect domain in the complex was located by molecular replacement with the program AMORE (39). Difference Fourier maps had clear electron density for the portion of UbcH7 that binds the hect domain. The UbcH7 structure was built using the Ubc4 structure (23) as a guide, and the structure of the complex was refined at 2.6 Å (Table 1) with the program CNS (41). The refined model contains residues 4 to 147 of UbcH7; residues 1 to 3 and 148 to 154 have no clear electron density. In the crystals, UbcH7 makes only a small number of crystal-packing contacts, and the portion of the UbcH7 structure distal from the hect-interacting portion has high temperature factors in the refined model.
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    • 727) making key trimer contacts has little effect on the ubiquitinthioester bond formation (L. Huang, E. Kinnucan, G. Wang, S. Beaudenon, J. M. Huibregtse, N. P. Pavletich, data not shown). The interactions associated with the formation of the trimer could, in principle, have favored the crystallization of the E6AP-UbcH7 complex in the same E6AP conformation as in the E6AP-alone crystals.
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    • 32P-labeled ubiquitin, and ATP. Reactions were incubated at room temperature for 5 to 10 min and stopped with SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer lacking reducing agent. Products were analyzed by SDS-PAGE and autoradiography and quantified with a Molecular Dynamics Phosphoimager. The amounts of ubiquitinthioester bond formation for the mutants, relative to the wild-type protein, are 0% for C820S, 4% for H818A, 26% for T819A, 31% for F821A, 28% for N822A, 8% for R506A, 7% for E539A, 6% for E550A, and 10% for D607A. Each reaction was repeated three times, with typical error bars of up to 4% of the wild-type activity.
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    • 2-terminal sequence analysis; C. Ogata of the National Synchrotron Light Source (NSLS) X4A beamline, M. Sullivan of the NSLS X9B beamline, and T. Earnest, C. McDermott, K. Henderson, and L. Hung at the Advanced Light Source Beamline 5.0.2 for help with data collection; P. Jeffrey for help with synchrotron data collection; and C. Murray for administrative assistance. Coordinates have been deposited with the Protein Data Bank (accession codes 1D5F for E6AP and 1C4Z for the E6AP-UbcH7 complex).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.