Cryo‐EM structure of the full‐length Lon protease from <i>Thermus thermophilus</i>

Coscia, Francesca; Löwe, Jan

doi:10.1002/1873-3468.14199

Cited by 10 publications

(8 citation statements)

References 59 publications

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“…Remarkably, data collection and image analysis yielded the structure of PIM1 in a hexameric, closed, substrate-translocating state at a reported resolution of ∼3.2 Å. Notably, and in stark contrast to all prior cryo-EM studies of Lon proteases in other species 13,14,[16][17][18] , we did not observe the presence of a fully ADP-bound substrate-free oligomer, typically defined by the AAA+ domains adopting an open, left-handed spiral organization. We note a high degree of background in our micrographs, which we believe to be monomeric PIM1 derived from biochemical instability of the hexamer or particle disruption at the air-water interface (Supplementary Figure 2A).…”

Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementscontrasting

confidence: 60%

“…Prior structural studies of substrate-translocating AAA+ proteins and Lon proteases are consistent with this model of hand-over-hand substrate translocation 13,14,[16][17][18] . In this model, pore loop residues of ATP-bound subunits form a spiral arrangement wherein the lower-most subunit's pore loop residue typically exists in an intermediary state.…”

Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementssupporting

confidence: 59%

“…In this model, pore loop residues of ATP-bound subunits form a spiral arrangement wherein the lower-most subunit's pore loop residue typically exists in an intermediary state. In concordance with the AAA+ organization of bacterial Lon proteases 13,18,19 , our substrate-translocating PIM1 structure consists of four descending ATPase domains (labeled here as ATP1-3, ATP4) and two ascending 'seam' subunits (ADP1-2). The ADP-bound subunits are found between the lowest and highest subunits of the substrate-bound spiral arrangement and ADP-bound subunit pore loop residues are indeed disengaged from substrate (Figure 1B, ADP1/ADP2).…”

Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementsmentioning

confidence: 76%

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Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Yang

Song

Wiseman

et al. 2021

Preprint

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Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a ~50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a ~15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1’s enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

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Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementscontrasting

confidence: 60%

Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementssupporting

confidence: 59%

Section: Substrate-bound Structure Of Pim1 Demonstrates Hexameric Assembly and Highlights Conserved Structural Elementsmentioning

confidence: 76%

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Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Yang

Song

Wiseman

et al. 2021

Preprint

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“…At first glance, the organization resembles that of Lon proteases, in which NTD dimers crown a homohexameric ATPase ring, resulting in a “trimer-of-dimers” arrangement 60 , 73 , 74 . However, the NTD dimers in Pex1/Pex6 are heterodimers formed by complementary charges between the domains of two different proteins (Fig.…”

Section: Discussionmentioning

confidence: 99%

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

Rüttermann,

Koci,

Lill

et al. 2023

Nat Commun

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The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a “twin-seam” Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.

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“…In Escherichia coli , more than half of the intracellular proteolysis is carried out by Lon protease [ 5 ]. The bacterial Lon protease (LonA) consists of three major domains: the N-terminal domain (NTD) that oligomerizes and recognizes substrates, the hexameric AAA+ (A) domain with unfolding activity, i.e., ATP binding and hydrolysis, and the C-terminal serine protease (P) domain, which hydrolyses substrates [ 6 , 7 ]. In the absence of a substrate, the enzyme’s hexameric ring adopts an open conformation.…”

Section: Introductionmentioning

confidence: 99%

Structure, Substrate Specificity and Role of Lon Protease in Bacterial Pathogenesis and Survival

Kirthika

Lloren

Jawalagatti

et al. 2023

IJMS

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Proteases are the group of enzymes that carry out proteolysis in all forms of life and play an essential role in cell survival. By acting on specific functional proteins, proteases affect the transcriptional and post-translational pathways in a cell. Lon, FtsH, HslVU and the Clp family are among the ATP-dependent proteases responsible for intracellular proteolysis in bacteria. In bacteria, Lon protease acts as a global regulator, governs an array of important functions such as DNA replication and repair, virulence factors, stress response and biofilm formation, among others. Moreover, Lon is involved in the regulation of bacterial metabolism and toxin–antitoxin systems. Hence, understanding the contribution and mechanisms of Lon as a global regulator in bacterial pathogenesis is crucial. In this review, we discuss the structure and substrate specificity of the bacterial Lon protease, as well as its ability to regulate bacterial pathogenesis.

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Cryo‐EM structure of the full‐length Lon protease from Thermus thermophilus

Cited by 10 publications

References 59 publications

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

Structure, Substrate Specificity and Role of Lon Protease in Bacterial Pathogenesis and Survival

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