Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release.
The highly conserved, 300-kDa cylindrical protease ClpP is an important component of the cellular protein quality machinery. It consists of 14 subunits arranged into two heptameric rings that enclose a large chamber containing the protease active sites. ClpP associates with ClpX and ClpA ATPases that unfold and translocate substrates into the protease catalytic chamber through axial pores located at both ends of the ClpP cylinder. Although the pathway of substrate delivery is well established, the pathway of product release is unknown. Here, we use recently developed transverse relaxation optimized spectroscopy (TROSY) of methyl groups to show that the interface between the heptameric rings exchanges between two structurally distinct conformations. The conformational exchange process has been quantified by magnetization exchange and methyl TROSY relaxation dispersion experiments recorded between 0.5 degrees C and 40 degrees C, so that the thermodynamic properties for the transition could be obtained. Restriction of the observed motional freedom in ClpP through the introduction of a cysteine linkage results in a protease where substrate release becomes significantly slowed relative to the rate observed in the reduced enzyme, suggesting that the observed motions lead to the formation of transient side pores that may play an important role in product release.
Sprangers
Remco
R
Department of Biochemistry, University of Toronto, ON, Canada.
Gribun
Anna
A
Hwang
Peter M
PM
Houry
Walid A
WA
Kay
Lewis E
LE
D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp