The barrel-shaped serine protease ClpP degrades misfolded, damaged, and regulatory proteins. Substrate proteins enter the ClpP barrel through the two axial pores, but it is unclear how the peptide products exit the barrel. Here we report the structure of ClpP from Staphylococcus aureus, which reveals a previously unobserved compressed state of the barrel. A conformational switch in the active center "handle region" results in closure of the active sites and opening of equatorial pores. Conserved residues in the handle region underlie the conformational switch and are functionally essential although they are not part of the active sites. These results are consistent with processive cycling of ClpP between an extended state with open active sites and closed equatorial pores, and a compressed state with closed active sites and open pores for product release.
Energy-dependent proteases control quality and quantity of cellular proteins by degrading those that are misfolded, damaged, or regulatory and thus short-lived. [1] Proteases of this family include the eukaryotic 26S proteasome and the bacterial caseinolytic protease (Clp). [2a,b] Clp enzymes contain the barrel-shaped serine protease ClpP that degrades the substrate protein, and an AAA + ATPase such as ClpX or ClpA that selects and unfolds the substrate and translocates it into the ClpP barrel through its axial pores. [3a,b] ClpP processively digests the translocated polypeptide into peptide fragments of six to eight residues. [4] Crystal structures of ClpP from various species are highly similar and reveal two stacked heptameric rings, which confine a large chamber containing 14 active sites. [5] ClpP is a serine protease with canonical catalytic triads that each comprise a serine, histidine, and aspartate residue. Structures of ClpP bound to an inhibitor [6] or a peptide [7] revealed that the active center region is flexible and flanked by a mobile "handle region" that is functionally important. [8,9] The key open question on the ClpP mechanism is how the peptide products are released from the active sites and how they exit from the barrel chamber. One model proposes that the peptides diffuse through the axial pores that are also used for substrate entry, [10a,b] but this model is inconsistent with published data [9] and postulates transient release of the ATPases from ClpP and thus seems incompatible with a processive function. [11] A second model proposes that the two heptameric rings dissociate transiently, but there is no evidence for this. A third model proposes that peptides are released through "equatorial" pores that transiently form on the side of the barrel. [8,9,12] However, such pores have not been observed and their formation would require major conformational changes that seem difficult without barrel dissociation.
Here we report the crystal structure of ClpP from Staphylococcus aureus (SaClpP), which reveals the predicted equatorial pores and the conformational changes required for their formation without barrel dissociation.
SaClpP was purified and crystallized, and its structure determined by molecular replacement using ClpP from Streptococcus pneumoniae (SpClpP, 1Y7O) [8] as a search model (see Table 1 and the Supporting Information). Like ClpP from other species, [5] SaClpP consists of two heptameric rings that form a tetradecameric barrel (Figure 1). However, compared to all other ClpP structures, the barrel is compressed by roughly 10 along the axial direction, and the ring-ring interface contains 14 equatorial side pores that can be up to 6 in diameter, depending on the side-chain conformations (Figure 2). The side pores are lined with conserved hydrophobic residues and apparently represent the long-sought exit route for peptide products from the barrel chamber. [8,9] The compressed barrel reflects a different conformational state rather than a species-specific fold, since SaClpP shares 73 % identical residues with SpClpP, and is highly conserved throughout species.