Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis

Abstract

The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.

Keywords: ClpP; acyldepsipeptide; cryo-EM; pH drop; protein degradation.

Figure EV1. Sequence alignment among ClpPs

The UniProt IDs for the ClpP sequences are Bacillus subtilis ClpP (BsClpP) P80244, Neisseria meningitidis ClpP (NmClpP) Q9JZ38, Mycobacterium tuberculosis ClpP1 (MtuClpP1) P9WPC5, Mycobacterium tuberculosis ClpP2 (MtuClpP2) P9WPC3, and Staphylococcus aureus ClpP (SaClpP) Q2G036. Secondary structure elements are indicated (α‐helices ‐ coil; β‐strands ‐ arrow) and labeled above the sequence. Filled blue circles indicate the catalytic triad (S97‐H122‐D171), and the flexible handle region is marked with a black dashed box. His145 in NmClpP and its equivalent residues are highlighted in yellow. Identical residues are shaded in red, and highly conserved residues are marked in blue boxes. The sequence number for B. subtilis ClpP is indicated above the sequence.

Figure 1. pH change during protein hydrolysis

1. Monitoring of the pH change during the degradation of α‐casein by proteinase K and BsClpP in the presence of ADEP1.

2. Monitoring of the pH change during the degradation of bovine serum albumin (BSA) by proteinase K.

3. Monitoring of the pH change during the degradation of myoglobin by proteinase K.

4. Monitoring of the pH change during the degradation of the BsFtsZ‐ED6K mutant by proteinase K and BsClpP in the presence of ADEP1.

Data information: In all panels (A–D), black circles represent the control, only substrates without proteases. Red (triangle) and blue (square) symbols represent proteinase K and BsClpP+ADEP1, respectively. Data points represent the mean value of three measurements, and the error bars show the standard deviations.

Figure EV2. SDS–PAGE results showing protein hydrolysis in the course of the pH‐drop experiment presented in Fig 1

1. A

   Substrates α‐casein (red arrowhead) and bovine serum albumin (BSA: black arrowhead) were incubated with proteinase K (ProK: white arrowhead).

2. B

   Substrate α‐casein (red arrowhead) was incubated with BsClpP (green arrowhead) in the presence of ADEP1.

3. C, D

   Substrate myoglobin (blue arrowhead) was incubated with (C) and without proteinase K (white arrowhead) (D).

4. E, F

   Basic BsFtsZ‐ED6K substrate (yellow arrowhead) was incubated with proteinase K (white arrowhead) (E) and BsClpP (green arrowhead) + ADEP1 (F).

Data information: In all panels (A–F), the samples were collected at each time point. The “M” lane indicates the molecular weight marker (240, 140, 100, 70, 50, 35, 25, 20, 15, 7 kDa as shown in panel (A)).

Figure 2. X‐ray structures of ADEP‐bound BsClpP

1. Ribbon diagram of 4 ADEP‐bound BsClpP (cyan) with a transparent molecular surface and one monomer in each heptameric ring, colored darker for clarity (2ADEP).

2. Ribbon diagram of 10 ADEP‐bound BsClpP (green) with a transparent molecular surface and one monomer in each heptameric ring, colored darker for clarity (5ADEP). Tetradecameric BsClpP is viewed along a 7‐fold molecular symmetry axis (upper), and the 2‐fold side view is observed by rotating 90° (lower). The bound ADEP molecules colored red are shown as stick models. The dimensions of the models are indicated. Two diameters are noted due to the asymmetric shape of the entrance pore.

Figure 3. Structural analyses of compressed 2ADEP and compact 5ADEP

1. Three different subunit environments of 2ADEP viewed along a 7‐fold axis: two cyanish subunits (1 and 1′) with the bound ADEP (pink molecular surface) on the right side, two orangish subunits (2 and 2′) with the bound ADEP on the left side, and three yellowish subunits (3, 3′ and 3′′) with no ADEP molecule.

2. Superposition of all 7 subunits in the heptameric ring of 2ADEP viewed by rotating panel (A) 45° about the horizontal axis. The invisible N‐terminal region, due to flexibility, is marked with a transparent oval, and the structurally dynamic handle region is marked with a dashed circle.

3. Superposition of cyanish subunits (1, 1′), orangish subunits (2, 2′), and yellowish subunits (3, 3′, 3′′). The view is the same as that of panel (B).

4. Three different subunit environments of 5ADEP viewed along a 7‐fold axis: three greenish subunits (1, 1′ and 1′′) with the bound ADEP (pink molecular surface) on both the left and right sides, two bluish subunits (2 and 2′) with the bound ADEP on the left side, and two reddish subunits (3 and 3′) with the bound ADEP on the right side.

5. Superposition of all 7 subunits in the heptameric ring of 5ADEP viewed by rotating panel (D) 45° about the horizontal axis. The very flexible N‐terminal region and handle region are marked with dashed circles.

6. Superposition of greenish subunits (1, 1′, 1′′), bluish subunits (2, 2′), and reddish subunits (3, 3′). The view is the same as that of panel (E).

Figure EV3. Structural analyses of 2ADEP and 5ADEP related to Fig 3

1. Side view of the 2ADEP structure obtained by rotating the top view (Fig 3A) 90° about the horizontal axis (middle). Left and right, approximately 120° (counterclockwise (−) and clockwise (+)) rotation along the vertical axis showing asymmetric side pores. The side exit pores are marked with transparent red ovals. The colors and numbers indicating the subunit environments are the same as those in Fig 3A.

2. Side view of the 5ADEP structure obtained by rotating the top view (Fig 3D) 90° about the horizontal axis (middle). Left and right, the view is the same as that of panel (A). The colors and numbers indicating the subunit environments are the same as those in Fig 3D.

Figure 4. Cryo‐EM structures of the BsClpP‐ADEP1 complex

1. Ribbon diagram, with the transparent molecular surface of the extended apo‐BsClpP structure at pH 6.5 viewed along a 7‐fold molecular symmetry axis (upper row) and viewed with 90° rotation to display the side view of the 2‐fold symmetry axis (lower row). One monomer is shown in darker orange color for clarity.

2. Same representation as that of panel (A) showing the 14 ADEP1‐bound BsClpP at pH 6.5. The bound ADEP1 molecules are shown as stick models colored red.

3. Same representation as that of the compressed BsClpP with 14 ADEP1 molecules at pH 4.2. One monomer is shown in darker yellow color for clarity.

4. Same representation as that of the compressed apo‐BsClpP at pH 4.2. The dimensions of the models are indicated.

Figure EV4. Cryo‐EM data processing workflow

1. A–C

   Representative electron micrograph, 3‐D classification and map refinement workflow of the apo form of BsClpP at pH 6.5 (A), 14 ADEP‐bound form of BsClpP at pH 6.5 (B), and apo and 14 ADEP‐bound forms of BsClpP at pH 4.2 (C).

2. D

   Map resolution for each BsClpP cryo‐EM structure according to the gold‐standard Fourier shell correlation (FSC) at the 0.143 criterion.

Figure 5. Electrostatic potential of the proteolytic chamber of various structural states

1. Ribbon diagram, with the transparent electrostatic potential surface of the extended apo‐BsClpP viewed from the center of the proteolytic chamber. The catalytic triad (S97‐H122‐D171) is shown in the fully active configuration.

2. Same representation for the 14 ADEP‐bound BsClpP cryo‐EM structure at pH 6.5. The residues maintaining the extended conformation are now slightly loosened, and the catalytic triad is slightly distorted.

3. Same representation for the 14 ADEP‐bound BsClpP at pH 4.2. The conformation is now compact, and side exit pores (black arrows) start to appear.

4. Same representation for 5ADEP at pH 5.6. The conformation of BsClpP is compact, and the catalytic triads in the upper and lower heptameric rings become closer and exhibit an inactive configuration.

5. Same representation for 2ADEP at pH 4.2. The conformation of BsClpP is compressed, and the catalytic triads in the upper and lower heptameric rings are very close.

6. Same representation for the compressed apo‐BsClpP. The exit pores are wide open for product release.

Data information: In all panels (A–F), the residues maintaining the extended conformation of ClpP are shown as stick models and labeled. For clarity, the residues in the neighboring subunits of the different heptameric rings are marked with prime (′) after the residue number, and those in the neighboring subunits of the same heptameric ring are marked with prime (′) before a single letter code used to represent the amino acid. Positively and negatively charged surfaces are colored blue and red, respectively. The inner surface of the proteolytic chamber is virtually negatively charged (red color), and the acidic proteolytic products must be energetically very unfavorable.

Figure EV5. Comparison of the activator binding sites of ClpP

1. Overall view of two conformations (6W21 in cyan and 6W20 in yellow) of the E. coli ClpAP complex. The ClpAP complex was superimposed with BsClpP at pH 6.5 (orange) complexed with ADEP (pink). The red boxed region indicates the interaction region between ClpP and the activators ClpA or ADEP.

2. Six IGL loops of ClpA overlap with the ADEP molecules, and thus, only one site of ClpP is empty in this conformation of the ClpAP complex (cyan). Enlarged view of the red boxed region in (A).

3. Only five IGL loops of ClpA overlap with the ADEP molecules; thus, two sites of ClpP are empty in this conformation of the ClpAP complex (yellow). The binding sites left empty by ClpAP are marked with white dotted circles in ClpAP, whereas ADEP occupies these sites in the ADEP‐BsClpP structure. The conformation of ClpAP in panel (C) shows slightly more tilted angles (14 versus 16°), as shown in panel (A) (Lopez et al, 2020). Enlarged view of the red boxed region in (A).

Figure 6. Model proposed for product release of ClpP

Before (state 1) and immediately after substrate binding (state 2), ClpP is in an active extended conformation, and ADEP widens the entrance pore for better substrate feeding. Our previous crystal structure of the 14 ADEP complex showed a disordered N‐terminal region (Lee et al, 2010), and our current cryo‐EM structure of the same complex demonstrates an ordered N‐terminal region. In both cases, the entrance pore is enlarged. From the extended structure (states 1 and 2) to the compressed structure (states 5 and 6), intermediate conformations were observed. Based on the solution cryo‐EM analysis, these intermediate conformations represented heterogeneous complex species of BsClpP with different numbers of ADEP molecules bound, including a 14 ADEP‐bound compact structure (state 3). Two major species among the intermediate states, 5ADE P (state 4) and 2ADEP (state 5), were crystallized and showed dimensions similar to those of previously known “compact” and “compressed” structures (Geiger et al, ; Lee et al, ; Zhang et al, ; Gersch et al, 2012). Both possess asymmetric entrance pores, especially 5ADEP, which is more distorted. 5ADEP is less compressed, and thus, the number and size of the side pores are fewer and smaller than those of a fully compressed structure, respectively. Apo‐BsClpP at pH 4.2 exhibits compression and larger side exit pores (state 6). The extended states 1 and 2 possess the active catalytic triad configuration (Fig 5A and B). The catalytic triad of the other states (3–6) shows an inactive and distorted configuration (Fig 5C–F). In the model, the red color of the proteolytic chamber represents the lower pH condition derived from substrate hydrolysis.