title: Dual stoichiometry and subunit organization in the ClpP1/P2 protease from the cyanobacterium Synechococcus elongatus.
abstract: 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×(3ClpP1+4ClpP2) with a low population of a 2×(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.
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK.
authors.forename: Victor A
authors.initials: VA
authors.lastname: Mikhailov
authors.affiliation: Department of Biological & Environmental Sciences, Gothenburg University, Gothenburg, Sweden.
authors.forename: Frida
authors.initials: F
authors.lastname: Ståhlberg
authors.affiliation: Department of Biological & Environmental Sciences, Gothenburg University, Gothenburg, Sweden.
authors.forename: Adrian K
authors.initials: AK
authors.lastname: Clarke
authors.affiliation: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, UK. Electronic address: carol.robinson@chem.ox.ac.uk.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000648:ultrastructure*; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D013058:Mass Spectrometry; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology*; D021122:Protein Subunits / Q000032:analysis*; D046940:Synechococcus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D049071:Endopeptidase Clp
keywords: Clp protease; Enzyme structure; Macromolecular protein complexes; Mass spectrometry (MS); Stoichiometry; Structural biology