Dual stoichiometry and subunit organization in the ClpP1/P2 protease from the cyanobacterium Synechococcus elongatus

作者：Mikhailov Victor A; Stahlberg Frida; Clarke Adrian K; Robinson Carol V^*

来源：Journal of Structural Biology, 2015, 192(3): 519-527.

DOI：10.1016/j.jsb.2015.10.016

• Clp protease
• Enzyme structure
• Macromolecular protein complexes
• Structural biology
• Mass spectrometry (MS)
• Stoichiometry

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. To investigate the proteolytic core of the ClpXP1/P2 protease from the cyanobacterium Synechococcus elongatus we have used a non-denaturing mass spectrometry approach. We show that the proteolytic core is a double ring tetradecamer consisting of an equal number of ClpP1 and ClpP2 subunits with masses of 21.70 and 23.44 kDa, respectively. Two stoichiometries are revealed for the heptameric rings: 4ClpP1 + 3ClpP2 and 3ClpP1 + 4ClpP2. When combined in the double ring the stoichiometries are (4ClpP1 + 3ClpP2) + (3ClpP1 + 4ClpP2) and 2 x (3ClpP1 + 4ClpP2) with a low population of a 2 x (4ClpP1 + 3ClpP2) tetradecamer. The assignment of the stoichiometries is confirmed by collision-induced dissociation of selected charge states of the intact heptamer and tetradecamer. Presence of the heterodimers, heterotetramers and heterohexamers, and absence of the mono-oligomers, in the mass spectra of the partially denatured protease indicates that the ring complex consists of a chain of ClpP1/ClpP2 heterodimers with the ring completed by an additional ClpP1 or ClpP2 subunit.

• 出版日期2015-12