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A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

ABSTRACT: Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.

SUBMITTER: Li S

PROVIDER: S-EPMC10643698 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

Li Shanshan S Hsieh Kan-Yen KY Kuo Chiao-I CI Lin Tzu-Chi TC Lee Szu-Hui SH Chen Yi-Ru YR Wang Chun-Hsiung CH Ho Meng-Ru MR Ting See-Yeun SY Zhang Kaiming K Chang Chung-I CI

Nature communications 20231113 1

Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show h ...[more]

PMID: 37957149

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Project description:Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during growth in standard aerobic conditions. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter.ImportanceThe Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due in part to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

Project description:Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus , low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address if the degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that Δ lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as there is diminished expression of many FixLJ-activated genes in Δ lon . However, no physiological changes in response to microaerobic conditions occurred upon loss of Lon, suggesting that FixT dynamics are not a major contributor to fitness in oxygen limiting conditions. Similarly, stabilization of FixT in Δ lon strains does not contribute to any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during adaptation to long term aerobic growth. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter .ImportanceThe Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

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A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

Publications

A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

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