Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis

Nagpal, Jyotsna; Paxman, Jason J.; Zammit, Jessica E.; Alhuwaider, Adnan Ali H.; Truscott, Kaye N.; Heras, Begoña; Dougan, David A.

doi:10.1038/s41598-019-53736-8

Cited by 17 publications

(25 citation statements)

References 53 publications

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“…This apparently contradicts previous evidence for the requirement of both ClpP1 and ClpP2 for assembly of the proteolytically active degradation compartment . Moreover, it is the ClpP2, rather than ClpP1, ring that binds ClpC1 in the context of the ClpCP complex . Noteworthy, our analysis was based on gene knockdown, rather than on gene deletion mutants, raising the possibility that clpP2 expression was not knocked down to levels sufficiently low in our study.…”

Section: Discussioncontrasting

confidence: 99%

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

et al. 2020

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Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a Pup-proteasome system tags target proteins for degradation by a bacterial proteasome. Pup, a small modifier protein, is attached to target proteins by PafA, the sole Pup ligase, in a process termed pupylation. In mycobacteria, including Mycobacterium smegmatis and Mycobacterium tuberculosis, Pup undergoes a deamidation step by the enzyme Dop prior to its PafA-mediated attachment to a target. The catalytic mechanism of Pup deamidation is also used by Dop to perform depupylation, namely the removal of Pup from already tagged proteins. Hence, Dop appears to play contradictory roles: On the one hand, deamidation of Pup promotes pupylation, while on the other hand, depupylation reduces tagged protein levels. To avoid futile pupylation-depupylation cycles, Dop activity must be regulated. An intramolecular regulatory mechanism directs Dop to catalyze deamidation more effectively than depupylation. A complementary intermolecular mechanism results in Dop depletion under conditions where protein pupylation and degradation are favorable. In this work, we studied these regulatory mechanisms and identified a flexible loop in Dop, previously termed the Dop-loop, that acts as an intramolecular regulatory element that allosterically controls substrate preference. To investigate regulation at the intermolecular level, we used the CRISPR interference system to knock down the expression of M. smegmatis ATP-dependent intracellular proteases and found that the ClpCP protease is responsible for Dop depletion under starvation conditions. These findings clarify previous observations and introduce a new level for the regulation of Dop activity.

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Section: Discussioncontrasting

confidence: 99%

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

et al. 2020

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“…Consortium (ID 3349399). For the heterologous expression of PDIP38 in E. coli , the cDNA coding for mature PDIP38 (residues 52–368) was amplified by PCR from pOTB7/ PDIP38 using the appropriate primers (Supplementary Table 2 ) and cloned into either pHUE 49 between SacII and HindIII (to express untagged PDIP38), pET10N 77 between NotI and XhoI (to express PDIP38 with an N-terminal H 10 tag), pET10C 77 between NdeI and NotI (to express PDIP38 as a C-terminal H 10 fusion protein), pGEX-4T-1 between BamHI and XhoI (to express PDIP38 as an N-terminal GST-fusion protein) or pDD173 78 between NotI and HindIII (to express PDIP38 as a C-terminal GFP fusion protein with an N-terminal H 10 tag). To generate PDIP38 N (residues 52–153) and PDIP38 C (residues 157–368) fused to GST, pGEX-4T/ PDIP38 was subjected to site-directed mutagenesis 79 using primers PDIP_bam1 and PDIP_bam2 (see Supplementary Table 2 ).…”

Section: Methodsmentioning

confidence: 99%

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP

et al. 2020

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Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/β linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like β-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like β-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

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“…However, both protease subunits were capable of comparable rates of model protein degradation, suggesting proteolytic redundancy in the Mtb ClpP1P2 complex (6,9,10). Despite this apparent redundancy in vitro, both ClpP1 and ClpP2 proteolytic activity are proposed to be necessary for Mtb growth and pathogenesis (9,11,12). Because of the historical technical challenges posed by studying essential Clp genes in mycobacteria, correlations between biochemical studies and in vivo function remain scarce (12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

The essential M. tuberculosis Clp protease is functionally asymmetric in vivo

et al. 2022

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The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli , the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.

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Molecular and structural insights into an asymmetric proteolytic complex (ClpP1P2) from Mycobacterium smegmatis

Cited by 17 publications

References 53 publications

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Inter‐ and intramolecular regulation of protein depupylation in Mycobacterium smegmatis

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP

The essential M. tuberculosis Clp protease is functionally asymmetric in vivo

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