STRUCTURE AND FUNCTION OF THE CLPXP ATP-DEPENDENT PROTEASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We had previously solved the structure of the AAA+ adaptor protein SspB in complex with one of its substrates, the ssrA tag. In E. coli, this tag targets incompletely synthesized proteins from stalled ribosomes for destruction by the ClpXP degradation machine, a process that is accelerated by SspB. Biochemical studies have revealed that other substrates are targeted to ClpXP by SspB, including the n-terminal fragment of RseA. RseA is a trans-membrane protein that functions as a master regulator of the extra-cytoplasmic stress response in E. coli. Targeting of n-terminal fragment for degradation by ClpXP is required for the release of the sigma factor (sigma E) and activation stress response genes. Using biochemical and biophysical techniques, a 31 amino acid stretch of the RseA sequence was identified as the SspB-binding determinant. In order to understand the structural basis for the SspB-RseA interaction, we crystallized the protein in complex with this 31 amino acid peptide. Energy-dependent proteases often rely on adaptor proteins to modulate substrate recognition. The SspB adaptor binds peptide sequences in the stress-response regulator RseA and in ssrA-tagged proteins and delivers these molecules to the AAA+ ClpXP protease for degradation. The structure of SspB bound to an ssrA peptide is known. Here, we reported the crystal structure of a complex between SspB and its recognition peptide in RseA in Nat. Struct. Mol. Biol. Notably, the RseA sequence is positioned in the peptide-binding groove of SspB in a direction opposite to the ssrA peptide, the two peptides share only one common interaction with the adaptor, and the RseA interaction site is substantially larger than the overlapping ssrA site. This marked diversity in SspB recognition of different target proteins indicates that it is capable of highly flexible and dynamic substrate delivery