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

Leehyeon Kim; Byung Gil Lee; Minki Kim; Min Kyung Kim; Do Hoon Kwon; Hyunmin Kim; Heike Brötz-Oesterhelt; Soung Hun Roh; Hyun Kyu Song

doi:10.15252/embj.2021109755

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

Leehyeon Kim, Byung Gil Lee, Minki Kim, Min Kyung Kim, Do Hoon Kwon, Hyunmin Kim, Heike Brötz-Oesterhelt, Soung Hun Roh, Hyun Kyu Song

Research output: Contribution to journal › Article › peer-review

9 Citations (Scopus)

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.

Original language	English
Article number	e109755
Journal	EMBO Journal
Volume	41
Issue number	13
DOIs	https://doi.org/10.15252/embj.2021109755
Publication status	Published - 2022 Jul 4

Bibliographical note

Funding Information:
We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X‐ray data collection. We also thank the staff of the cryo‐TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project‐ID 398967434 and cluster of excellence CMFI, project‐ID 390838134.

Funding Information:
We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X-ray data collection. We also thank the staff of the cryo-TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project-ID 398967434 and cluster of excellence CMFI, project-ID 390838134.

Publisher Copyright:
© 2022 The Authors.

Keywords

ClpP
acyldepsipeptide
cryo-EM
pH drop
protein degradation

ASJC Scopus subject areas

General Neuroscience
Molecular Biology
General Biochemistry,Genetics and Molecular Biology
General Immunology and Microbiology

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10.15252/embj.2021109755

Cite this

@article{b492d4269f094b8e8759a755aa30253b,

title = "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",

author = "Leehyeon Kim and Lee, {Byung Gil} and Minki Kim and Kim, {Min Kyung} and Kwon, {Do Hoon} and Hyunmin Kim and Heike Br{\"o}tz-Oesterhelt and Roh, {Soung Hun} and Song, {Hyun Kyu}",

note = "Funding Information: We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X‐ray data collection. We also thank the staff of the cryo‐TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project‐ID 398967434 and cluster of excellence CMFI, project‐ID 390838134. Funding Information: We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X-ray data collection. We also thank the staff of the cryo-TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project-ID 398967434 and cluster of excellence CMFI, project-ID 390838134. Publisher Copyright: {\textcopyright} 2022 The Authors.",

year = "2022",

month = jul,

day = "4",

doi = "10.15252/embj.2021109755",

language = "English",

volume = "41",

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T1 - Structural insights into ClpP protease side exit pore-opening by a pH drop coupled with substrate hydrolysis

AU - Kim, Leehyeon

AU - Lee, Byung Gil

AU - Kim, Minki

AU - Kim, Min Kyung

AU - Kwon, Do Hoon

AU - Kim, Hyunmin

AU - Brötz-Oesterhelt, Heike

AU - Roh, Soung Hun

AU - Song, Hyun Kyu

N1 - Funding Information: We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X‐ray data collection. We also thank the staff of the cryo‐TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project‐ID 398967434 and cluster of excellence CMFI, project‐ID 390838134. Funding Information: We thank the staff at beamlines 5C and 11C at the Pohang Accelerator Laboratory in South Korea and beamline NW12 at the Photon Factory in Japan for their help with X-ray data collection. We also thank the staff of the cryo-TEM facility at the Korea Basic Science Institute and the Center for Macromolecular and Cell Imaging, Seoul National University. This study was supported by National Research Foundation of Korea (NRF) grants from the Korean government (grant Nos. 2020R1A2C3008285, 2020R1A5A1019023, 2021M3A9G8024747, and 2021M3A9I4030068 for HKS; 2021M3A9I4021220, 2019M3E5D6063871, and 2020R1A5A1018081 for SHR; 2021R1A6A1A10045235 for LK) and the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) TRR 261, project-ID 398967434 and cluster of excellence CMFI, project-ID 390838134. Publisher Copyright: © 2022 The Authors.

PY - 2022/7/4

Y1 - 2022/7/4

N2 - 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.

AB - 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.

KW - ClpP

KW - acyldepsipeptide

KW - cryo-EM

KW - pH drop

KW - protein degradation

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ER -

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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