Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery

Botos, Istvan; Lountos, G.T.; Wu, Weimin; Cherry, Scott; Ghirlando, Rodolfo; Kudzhaev, A. M.; Rotanova, T. V.; Val, Natalia de; Tropea, Joseph E.; Gustchina, Alla; Wlodawer, Alexander

doi:10.1016/j.crstbi.2019.10.001

Cited by 25 publications

(29 citation statements)

References 43 publications

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“…Finally, we attempted to find out if the cytosolic domain of the asymmetric FtsH cryo-EM map possesses the staircase arrangement observed in other AAA ATPases (20,27) with the help of the high resolution structure of the YME1 protease (20). Its model (PDB ID: 6AZ0) describes an active state of YME1 during substrate processing, with four subunits bound to ATP, one to ADP, and one free of nucleotides, which gave rise to an asymmetric cytosolic domain (Figures 1, 4 and 5 in (20)).…”

Section: Cryo-emmentioning

confidence: 99%

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

Carvalho

Prabudiansyah

Kováčik

et al. 2021

Journal of Biological Chemistry

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AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore towards a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilisation induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ~20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible, and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.

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Section: Cryo-emmentioning

confidence: 99%

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

Carvalho

Prabudiansyah

Kováčik

et al. 2021

Journal of Biological Chemistry

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“…1 and Supplementary Table 1). The organization is reminiscent of the left-handed, open lockwasher configurations previously observed for substrate-free bacterial Lon 26,28 , although human LONP1 appears to oligomerize with a slightly smaller helical pitch than the bacterial forms (Fig. 1c,d and Supplementary Fig.…”

Section: Substrate-bound and Substrate-free Structures Of Human Lonpmentioning

confidence: 53%

“…The LONP1 protease consists of an N-terminal domain involved in substrate recognition and oligomerization, a AAA+ (ATPases associated with diverse cellular activities) domain that that powers substrate translocation, and a C-terminal serine protease domain involved in substrate proteolysis 1,4,18 . Numerous biophysical approaches have been employed to gain insight into the macromolecular structure and function of bacterial Lon [19][20][21][22][23][24][25][26][27] , and recent cryo-electron microscopy (cryo-EM) studies of bacterial Lon in both substrate-free and substrate-bound conformations revealed the structural basis for substrate processing and protease activation 28 . However, few structural studies have been directed towards understanding the molecular mechanism of protease activation and substrate processing by human LONP1.…”

Section: Introductionmentioning

confidence: 99%

Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

Shin

Watson

Novick

et al. 2020

Preprint

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The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in the pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the molecular mechanism of substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. However, unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate to increase interactions with the translocating peptide as it transits into the proteolytic chamber for proteolysis. Further, we show that substrate-bound LONP1 includes a second level of regulation at the proteolytic active site, wherein autoinhibition of the active site is only relieved by the presence of a peptide substrate. Ultimately, our results define a structural basis for human LONP1 proteolytic activation and activity, establishing a molecular framework to understand the critical importance of this protease for mitochondrial regulation in health and disease.

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“…For scoring parameters, BLOSUM62 matrix was used with the gap cost: Existence: 11 Extension: 1 with a threshold value of 0.005. In case of both the sequences, the A chain of PDB ID 6U5Z [14] showed the highest query coverage and percentage identity. In case of Uniprot ID P0A9M0 i.e.…”

Section: Sequence Homology Search and Modeling Of The Proteinsmentioning

confidence: 99%

Comparative analysis of the molecular mechanism of heat shock proteins from pathogenic and non-pathogenicE.coli

Bhattacharjee

Saha

Dasgupta

et al. 2020

Preprint

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Cells can withstand the effects of temperature stress by activating small heat shock proteins IbpA and IbpB. Lon protease employing Ser679 -Lys722 catalytic dyad proteolyze IbpA and IbpB in their free forms, at physiological temperature i.e. without any temperature stress. However, the proteolytic activity of IbpA and IbpB is affected when the catalytic dyad residue of Lon protease is mutated. The mutation S679A in Lon protease brings about some changes so that the proteolytic interactions between the small heat shock proteins with that of the mutant Lon protease are lost which makes a difference in the interaction pattern of mutant Lon protease with their substrates. In the present study, we made an attempt through in-silico approach to figure out the underlying aspects of the interactions between the small heat shock proteins IbpA and IbpB with mutant Lon protease in Escherichia coli. We have tried to decipher the molecular details of the mechanism of interaction of proteolytic machinery of small heat shock proteins and mutant Lon protease with S679A mutation at physiological temperature in absence cellular temperature stress. Our study may therefore be helpful to decode the mechanistic details of the correlation with IbpA, IbpB and S679A mutant Lon protease in E. coli.

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Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery

Cited by 25 publications

References 43 publications

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

Comparative analysis of the molecular mechanism of heat shock proteins from pathogenic and non-pathogenicE.coli

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