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.
Highlights
Bacterial caseinolytic protease P (ClpP) is a highly conserved, cylindrical, self-compartmentalizing serine protease required for maintaining cellular proteostasis
It is one of the main proteolytic complexes in bacteria[1]. It associates with hexameric unfoldase chaperones of the AAA+ (ATPases Associated with diverse cellular Activities) superfamily such as ClpX and ClpA in Escherichia coli that selectively bind, unfold, and translocate target proteins through the axial pores of ClpP and into the proteolytic chamber for degradation[2]
While a structure of NmClpP with bound ACP1 was not achieved despite repeated attempts, a structure of the EcClpP+ACP1-06 complex was determined to 1.9 Å resolution (Fig. 1b, c, Table 1), containing a tetradecamer in the asymmetric unit (Chains A-N)
Summary
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. The caseinolytic protease P (ClpP) is a tetradecameric serine protease comprised of two heptameric rings stacked together in a cylindrical structure It is one of the main proteolytic complexes in bacteria[1]. It associates with hexameric unfoldase chaperones of the AAA+ (ATPases Associated with diverse cellular Activities) superfamily such as ClpX and ClpA in Escherichia coli that selectively bind, unfold, and translocate target proteins through the axial pores of ClpP and into the proteolytic chamber for degradation[2]. Recent efforts in our laboratory to develop other ClpP dysregulators targeting Gram-negative bacteria have led to the discovery of a new class of potential antibiotics named as activators of self-compartmentalizing proteases 1 (ACP1s)[28].
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