ClpP protease activation results from the reorganization of the electrostatic interaction networks at the entrance pores

Abstract

Bacterial ClpP is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis. Small molecule acyldepsipeptides (ADEPs) and activators of self-compartmentalized proteases 1 (ACP1s) cause dysregulation and activation of ClpP, leading to bacterial cell death, highlighting their potential use as novel antibiotics. Structural changes in Neisseria meningitidis and Escherichia coli ClpP upon binding to novel ACP1 and ADEP analogs were probed by X-ray crystallography, methyl-TROSY NMR, and small angle X-ray scattering. ACP1 and ADEP induce distinct conformational changes in the ClpP structure. However, reorganization of electrostatic interaction networks at the ClpP entrance pores is necessary and sufficient for activation. Further activation is achieved by formation of ordered N-terminal axial loops and reduction in the structural heterogeneity of the ClpP cylinder. Activating mutations recapitulate the structural effects of small molecule activator binding. Our data, together with previous findings, provide a structural basis for a unified mechanism of compound-based ClpP activation.

Recent structural insights into the mechanism of ClpP protease regulation by AAA+ chaperones and small molecules

“…In the apo state, the N-terminal loops, also called axial pore loops, of ClpP block its axial pores and gate substrate entry (83). In many crystal structures, the binding of small molecule agonists to H sites (e.g., ADEPs) causes ordering of N-terminal loops into β-hairpin turns, resulting in an open gate conformation (83).…”

“…In the apo state, the N-terminal loops, also called axial pore loops, of ClpP block its axial pores and gate substrate entry (83). In many crystal structures, the binding of small molecule agonists to H sites (e.g., ADEPs) causes ordering of N-terminal loops into β-hairpin turns, resulting in an open gate conformation (83). This allosteric effect is also observed in the structure of NmClpXP, where Hydrogen-deuterium uptake of LmClpX IGF loops is reduced upon complex formation with LmClpP, suggesting solvent-shielding interactions with axial pore loops as also seen in NmClpXP structures (Figure 3C) (101).…”

Reprogramming of the Caseinolytic Protease by ADEP Antibiotics: Molecular Mechanism, Cellular Consequences, Therapeutic Potential

“…With the exception of human mitochondrial ClpP, which presented itself in a compact state despite ADEP binding (Wong et al, 2018), all ADEP-ClpP cocrystal structures reported to date show ClpP in the extended conformation with a widened entrance pore. This is the case for B. subtilis ClpP (Lee et al, 2010), E. coli ClpP (Li et al, 2010;Mabanglo et al, 2019), S. aureus ClpP (Vahidi et al, 2018;Griffith et al, 2019;Malik et al, 2020), Mycobacterium tuberculosis ClpP (Schmitz et al, 2014b;Vahidi et al, 2020), Neisseria meningitidis ClpP (Mabanglo et al, 2019), and Enterococcus faecium ClpP (Brown-Gandt et al, 2018). A critical residue for regulating the conformation of the N-terminal gate is a conserved tyrosine (e.g., Y63 in S. aureus and B. subtilis ClpP) within the H-pocket.…”

“…Another flexible domain of the ClpP protomer is the N-terminal loop. The seven N-terminal loops of a heptamer ring flank an entrance pore, and the dynamics of the N-terminal domain regulate pore diameter and thereby substrate access ( Alexopoulos et al, 2012 ; Vahidi et al, 2018 ; Mabanglo et al, 2019 ; Malik et al, 2020 ). Each apical face of ClpP presents seven cavities lined with hydrophobic amino acids, which serve as docking points for the Clp-ATPases.…”

“…Mutating the tyrosine to alanine has the same effect ( Ni et al, 2016 ). In the process of pore opening, the electrostatic interaction network at the ClpP entrance pores is reorganized, the normally flexible N-terminal loops lining the pore uniformly adopt an ordered β-hairpin conformation, and the entire N-terminal domain slightly moves outward ( Li et al, 2010 ; Alexopoulos et al, 2012 ; Schmitz et al, 2014b ; Mabanglo et al, 2019 ; Malik et al, 2020 ). The fact that ADEP binding to the H-pocket controls the conformation of the entire ClpP barrel is impressively visible in the ADEP-bound structure of M. tuberculosis ClpP ( Schmitz et al, 2014b ).…”