The degradation of ssrA(AANDENYALAA)-tagged proteins in the bacterial cytosol is carried out by the ClpXP protease and is markedly stimulated by the SspB adaptor protein. It has previously been reported that the amino-terminal zinc-binding domain of ClpX (ZBD) is involved in complex formation with the SspB-tail (XB: ClpX-binding motif). In an effort to better understand the recognition of SspB by ClpX and the mechanism of delivery of ssrA-tagged substrates to ClpXP, we have determined the structures of ZBD alone at 1.5, 2.0, and 2.5 Å resolution in each different crystal form and also in complex with XB peptide at 1.6 Å resolution. The XB peptide forms an antiparallel β-sheet with two β-strands of ZBD, and the structure shows a 1:1 stoichiometric complex between ZBD and XB, suggesting that there are two independent SspB-tail-binding sites in ZBD. The high-resolution ZBD:XB complex structure, in combination with biochemical analyses, can account for key determinants in the recognition of the SspB-tail by ClpX and sheds light on the mechanism of delivery of target proteins to the prokaryotic degradation machine.
Bibliographical noteFunding Information:
We thank the staff at 4A beamline, Pohang Accelerator Laboratory, Republic of Korea and NW12 beamline, Photon Factory, Japan for help with data collection. We also thank Drs P. Zwickl (Max-Planck-Institute for Biochemistry, Germany) and T. Tamura (National Institute of Advanced Industrial Science and Technology, Japan) for cells containing His-GFP-ssrA and Professor M. J. Eck for his generous support during the initial stage of this project. This work was supported by the Korea Research Foundation Grant (KRF-2006-312-C00249) funded by the Korean Government (MOEHRD) and by the Korea Science and Engineering Foundation Grant (KOSEF, R01-2004-000-10773-0) funded by the Korea government (MOST). E.Y.P. was supported by a Seoul Science Fellowship.
- ATP-dependent protease
- delivery complex
- ssrA tag
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology