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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.

SUBMITTER: Kim L

PROVIDER: S-EPMC9251834 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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

Kim Leehyeon L Lee Byung-Gil BG Kim Minki M Kim Min Kyung MK Kwon Do Hoon DH Kim Hyunmin H Brötz-Oesterhelt Heike H Roh Soung-Hun SH Song Hyun Kyu HK

The EMBO journal 20220520 13

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 mec ...[more]

PMID: 35593068

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Project description:The use of liposomal delivery systems for the treatment of cancer has been extensively researched because of their passive targeting to the vasculature of solid tumors. While their potential to provide prolonged retention and high drug encapsulation is desirable for anticancer agents, a mechanistic understanding is required to optimize and design liposomal drug delivery systems capable of controllable release tailored to tumor type and patient. Topotecan (TPT) is a topoisomerase I inhibitor that undergoes reversible, pH-sensitive ring-opening hydrolysis. TPT may benefit from liposomal formulation using active loading strategies to generate low intravesicular pH to prolong drug retention and increase drug encapsulation. This paper develops a mathematical model to describe TPT's permeability as a function of pH by accounting for the drug's ionization state, membrane binding, and ring-opening interconversion kinetics. Studies were conducted to determine the acid dissociation constant of TPT's phenolic -OH and interconversion kinetics between TPT's lactone and carboxylate forms. Using the constants determined from these studies and release studies conducted at varying pH, permeability coefficients and membrane binding constants for each species of TPT were determined. Based on this model, three permeable species were observed. Interestingly, the two most permeable species were zwitterionic forms of TPT, and the permeability of the lactone zwitterion was comparable to that of the neutral form of another camptothecin analogue. Furthermore, release was affected by based-catalyzed interconversion kinetics between TPT's lactone and carboxylate forms. At neutral pH, release was rate-limited by formation of the TPT lactone from the ring-opened carboxylate form. Based on these findings, the developed model describing liposomal release of TPT may be used in the future to evaluate and optimize loading and subsequent release of liposomal TPT formulations utilizing active loading strategies.

Project description:The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism. Factorial design comparing ClpP knock-out mice with wild type littermates in five different tissues (brain, testis, liver, skeletal and heart muscle)

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

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

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