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, 32 (6), 589-95

Structural Insights Into the Conformational Diversity of ClpP From Bacillus Subtilis

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Structural Insights Into the Conformational Diversity of ClpP From Bacillus Subtilis

Byung-Gil Lee et al. Mol Cells.

Abstract

ClpP is a cylindrical protease that is tightly regulated by Clp-ATPases. The activation mechanism of ClpP using acyldepsipeptide antibiotics as mimics of natural activators showed enlargement of the axial entrance pore for easier processing of incoming substrates. However, the elimination of degradation products from inside the ClpP chamber remains unclear since there is no exit pore for releasing these products in all determined ClpP structures. Here we report a new crystal structure of ClpP from Bacillus subtilis, which shows a significantly compressed shape along the axial direction. A portion of the handle regions comprising the heptameric ring-ring contacts shows structural transition from an ordered to a disordered state, which triggers the large conformational change from an extended to an overall compressed structure. Along with this structural change, 14 side pores are generated for product release and the catalytic triad adopts an inactive orientation. We have also determined B. subtilis ClpP inhibited by diisopropylfluoro-phosphate and analyzed the active site in detail. Structural information pertaining to several different conformational steps such as those related to extended, ADEP-activated, DFP-inhibited and compressed forms of ClpP from B. subtilis is available. Structural comparisons suggest that functionally important regions in the ClpP-family such as N-terminal segments for the axial pore, catalytic triads, and handle domains for the product releasing pore exhibit intrinsically dynamic and unique structural features. This study provides valuable insights for understanding the enigmatic cylindrical degradation machinery of ClpP as well as other related proteases such as HslV and the 20S proteasome.

Figures

Fig. 1.
Fig. 1.. Overall structure of ClpPs from B. subtilis and S. aureus. (A) Structure of ClpP from B. subtilis in the extended state shown as a ribbon diagram (top) and surface representation (bottom) viewed along a two-fold symmetry axis. Monomers are alternately colored green and sky-blue. (B) Structure of ClpP from B. subtilis in the compressed state with the same view and representation as in (A). Monomers are alternately colored yellow and orange. The location of two Clp-ATPases is shown as sky-blue ovals. (C) Structure of ClpP from S. aureus in the compressed state with the same view and representation as in (A). Monomers are alternately colored dark and bright gray. PDB IDs are provided. The approximate height of the molecules in this side view is indicated and the side pore utilized for product release is also shown as a red dotted circle.
Fig. 2.
Fig. 2.. Superposition of compressed and extended ClpP structures. (A) Compressed (orange) and extended (green) BsClpP monomer. The Nand C-terminus are labeled. Large conformational differences are found in the handle region, especially within α5-helix and β6-strand regions. (B) Close-up view of the region representing the greatest difference between compressed (orange) and extended (green) BsClpP. (C) Closeup view of the region representing the greatest difference between compressed BsClpP (orange) and SaClpP (slate). Invisible portions of the structures are shown as dots in all panels.
Fig. 3.
Fig. 3.. Active site of ClpPs. (A) Comparison of catalytic triad of compressed (orange) and extended (green) BsClpP. Ser97, His122 and Asp171 are shown as stick models with nitrogen and oxygen atoms in blue and red, respectively. The H-bonding distances between side chain atoms of Ser97 and His122 in two different structural states are indicated. (B) Structure of DFPinhibited BsClpP superposed with that of extended BsClpP (left) and its corresponding electron density for the DFP-inhibited covalently modified molecule (right). H-bonding distances are indicated.
Fig. 4.
Fig. 4.. Structural comparison of 4 different states of ClpP. (A) Molecular surface representation of the extended BsClpP structure viewed along a sevenfold molecular symmetry axis (top) and 90° rotation to display the side two-fold symmetry axis (bottom). The colors are the same as in Fig. 1A and one monomer is shown as a dark green ribbon for clarity. (B) The same representation as with ADEP1-bound BsClpP. Monomers are alternately colored red and purple, with one monomer shown as a dark red ribbon and bound ADEP1 molecules are shown as stick models with carbon, nitrogen and oxygen atoms in yellow, blue and red, respectively. (C) The same representation as with DFP-inhibited BsClpP. Monomers are alternately colored white and slate, with one monomer shown as a grey ribbon. (D) The same representation as with the compressed BsClpP structure. The colors are the same as in Fig. 1B and one monomer is shown in dark orange. The N-terminal β-1 strand is colored apricot for clarity in (A) and (C). PDB IDs are provided for each different structural state.

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