Structural insights into the Clp protein degradation machinery

Abstract

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Keywords: AAA+ ATPase; ADEP1; ClpP protease; protein degradation; substrate translocation mechanism.

Fig 1

Overview of the ClpC1:shClpP1P2 complex. (A) Linear representation of domain organization of shClpP1P2 and ClpC1. (B–C) Overall architecture of the substrate-bound ClpC1:shClpP1P2 complex. The side view (left) and top view (right) are shown. The ClpC1 protomers are individually colored. (D) Cross section of ClpC1:shClpP1P2 with a polypeptide substrate engaged by ClpC1 in the central channel.

Fig 2

Structures of apo-shClpP1P2 and the ADEP1:shClpP1P2 complex. (A–B) The tetradecameric structures of apo-shClpP1P2 (A) and ADEP1:shClpP1P2 (B). The ADEP1 molecules are shown as magenta sticks. (C) The intermolecular interactions between shClpP1 and shClpP2 stabilize the ring-ring interface of shClpP1P2. The hydrogen bonds and salt bridges are indicated by the dash lines between the interactive residues in the enlarged diagrams. (D) Apical hydrophobic pockets formed by two adjacent protomers of shClpP1 (left) or shClpP2 (right). The hydrophobic pockets are covered by the C-terminus of shClpP1 or shClpP2 (enclosed by dashed ovals) in the apo form (upper panel). Upon binding of ADEP1 (in magenta), the C-termini are expelled from the hydrophobic pockets (bottom panel). The key secondary structural elements are labeled.

Fig 3

Interaction between the LGF loop of ClpC1 and the hydrophobic pocket of shClpP2 in conformation A and B. (A) The bound ClpC1 LGF loop is shown in the cryo-EM density contoured at 3σ. (B) Superimposition of the LGF loops of the ClpC1 protomers in conformation A or B. The aromatic residues Phe677 are shown in sticks for figure clarification. (C) The LGF loop of ClpC1 at the P1 position is docked onto the shClpP2 surface in conformation B but undocked in conformation A. (D) The LGF loop of ClpC1 at the P3 position is bound within a hydrophobic pocket on the shClpP2 surface and stabilized by the extending C-terminus of shClpP2. (E) The C-terminal structures of the seven shClpP2 subunits are superimposed, indicating different conformations of the C-termini of shClpP2.

Fig 4

Substrate engagement by the ClpC1 pore loops. Comparison of conformation A and B reveals different substrate engagement states of the six ClpC1 protomers (P1–P6) in the D1 ring (left panel A and B) and the D2 ring (right panel C and D). The substrate engagement is shown in both the top views (upper panel A and C) and side views (bottom panel B and D). The polypeptide substrate is represented in magenta, and the six ClpC1 protomers are individually colored. The key tyrosine residues for substrate interaction, including Tyr257 of the D1 pore loop and Tyr597 of the D2 pore loop, are shown along the bound substrates, respectively.

Fig 5

Schematic of the ClpC1 nucleotide states in the two conformations and hypothetical mechanistic model for the substrate degradation by the ClpC1:shClpP1P2 machinery. (A–B) Schematic representations of the nucleotide states in the D1 ring (A) and the D2 ring (B) of ClpC1 in conformation A and conformation B. (C) Assembly of the functional Clp system in Streptomyces. (D) A proposed pathway for the ClpC1 rotation on the shClpP2 surface that is associated with the substrate transportation from the ClpC1 D1/D2 rings to the shClpP1P2 degradation chamber. The shClpP2 protomers are labeled clockwise from H to N according to their chain positions.