Structure and Function of a Novel Type of ATP-dependent Clp Protease

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. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP3 3 ClpR 4 configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.

Subunit Stoichiometry, Evolution, and Functional Implications of an Asymmetric Plant Plastid ClpP/R Protease Complex in Arabidopsis

“…The lower concentration of catalytic sites in the P1-R ring is likely to show a lower rate of proteolysis, which may or may not be the rate-limiting step in the sequence of substrate selection, delivery, and unfolding. In cyanobacteria, the inclusion of the catalytically inactive ClpR in the ClpP3/R complex appeared not to be rate limiting because restoration of the catalytic sites and replacement of the whole internal domain of ClpR with that of ClpP3 did not enhance the proteolytic activity (Andersson et al, 2009). Asymmetric Clp protease core particles derived from combinations of proteolytically inactive (mutation in catalytic site of ClpP) and chaperone binding-incompetent (N-terminal mutation) Clp rings from E. coli have been assembled in vitro (Maglica et al, 2009).…”

“…Intriguingly, the 3:4 active:inactive composition is also observed in heptameric component rings of the eukaryotic proteasome (Groll et al, 1997), suggesting a functional restraint on the composition of these various proteolytic assemblies. Intact MS analysis of the dissociated subcomplexes of the cyanobacterial ClpP3/R core revealed mainly heterodimers and other heteromeric combinations, indicating that the ClpP3 subunit is localized between two ClpR subunits within the ring (Andersson et al, 2009). This organization might also apply to the chloroplast-localized ClpP1-R ring, with the ClpP1 arranged between two ClpR subunits.…”

“…Photosynthetic bacteria possess three ClpP proteins (ClpP1-P3) and one ClpR protein, which is structurally similar to ClpP but lacks the catalytic residues for peptide bond hydrolysis (Schelin et al, 2002). These cyanobacterial proteins assemble into an essential ClpR/P3 complex and a nonessential ClpP1/P2 complex Andersson et al, 2009). …”

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation

“…3C). The Clp protease is a key component of the bacterial response to stress and is one of the major cellular proteases responsible for the selective degradation of misfolded proteins under stress (43,44). It is expressed in MTB under both aerated (45) as well as stressed conditions such as elevated temperatures and phagocytosis by macrophage (46).…”

Polydispersity as a Parameter for Indicating the Thermal Stability of Proteins by Dynamic Light Scattering

“…This development has helped to elucidate the critical relationship between protein conformation and function. [1][2][3][4] Many proteins change their structures and stability on binding to ligands: surface plasmon resonance (SPR) is one of the most promising methods for evaluating such protein-ligand interactions. [5][6][7][8][9] This method is useful for determination of the binding constant (KD), and the binding kinetics of protein-ligand interactions in flow systems on solid surfaces.…”