Heterosubunit Composition and Crystal Structures of a Novel Bacterial M16B Metallopeptidase

Maruyama, Yukie; Chuma, A.; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

doi:10.1016/j.jmb.2011.01.038

Cited by 18 publications

(19 citation statements)

References 46 publications

(56 reference statements)

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“…Crystal structures are available for all three subfamilies: M16A (e.g., IDE and pitrilysin), M16B (e.g., MPP and cytochrome bc1 complex core), and M16C (e.g., PreP and falcilysin) (Fig. S1) (7,13,14,(16)(17)(18)(19). All M16 proteases contain two homologous ∼50-kDa domains, one of which contains a conserved HXXEH zinc ion-binding motif.…”

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confidence: 99%

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

McCord¹,

Liang²,

Dowdell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ∼18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N-and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides.M16 metalloprotease | X-ray crystallography | substrate recognition P roteins in living organisms face acute and chronic challenges to their integrity, which necessitate proteostatic processes to protect their functions (1). Protein-protease networks play a key role in proteostasis by ensuring proper protein function through protein turnovers (2). Amyloidogenic peptides, such as amyloid β (Aβ) and amylin, present a major challenge to proteostasis, because they can form toxic aggregates that impair diverse physiological functions and contribute to human diseases (3, 4). Insulin-degrading enzyme (IDE), a Zn 2+ -metalloprotease, prefers to degrade amyloidogenic peptides to prevent the formation of amyloid fibrils (3). Exemplary substrates of IDE are insulin and Aβ, which are critical for the development of type 2 diabetes mellitus (DM2) and Alzheimer's disease (AD), respectively. Genetic analyses strongly support functional roles of IDE in the clearance of insulin and Aβ (2, 3). In humans, several single nucleotide polymorphisms at the IDE locus on human chromosome 10q are associated with DM2 and late-onset AD (5, 6).Structural analyses have provided significant insights to substrate recognition and catalysis by IDE. IDE has two ∼50-kDa αβαβα N-terminal (IDE-N) and C-terminal (IDE-C) halves, which are linked by a short hinge loop...

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confidence: 99%

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

McCord¹,

Liang²,

Dowdell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…MPP-like proteins are also present in bacteria [22] and the α-proteobacterial processing peptidase of Rickettsia prowazekii (RPP) is thought to be the closest modern example for the progenitor of MPP [23]. Several other bacterial peptidases have recently been described [24,25], including an example of the heterodimeric M16 peptidase from Sphingomonas sp. [26]. This one will be discussed in more detail below.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the deletion of only one amino acid may change the conformation of GRL itself and thus “close the entrance” to the MPP active site. The normal, partially closed conformation of WT MPP is not affected by the presence or absence of a substrate in its active site, unlike Sphingomonas sp. M16 peptidase, the peptidase that lacks a GRL, which adopts distinct closed and open conformations, depending on whether there is or is not a substrate bound to its active site [26]. …”

Section: Discussionmentioning

confidence: 99%

A Computational Study of the Glycine-Rich Loop of Mitochondrial Processing Peptidase

et al. 2013

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An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.

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“…To distinguish plant MPP subunits from cp1/cp2, we designate herein the former as cb 1 -α-MPP and cb 1 -β-MPP. It has been proposed that MPP evolved from a single subunit of a bacterial protease, followed by gene duplication and specialization that gave rise to a heterodimeric enzyme in mitochondria (Kitada et al 2007), although more recent studies suggested that the heterodimeric protease was formed already in bacteria (Maruyama et al 2011). Subsequently, another gene duplication resulted in the appearance of the membrane-associated core proteins and soluble MPP subunits.…”

Section: Introductionmentioning

confidence: 99%

An Advanced System of the Mitochondrial Processing Peptidase and Core Protein Family in Trypanosoma brucei and Multiple Origins of the Core I Subunit in Eukaryotes

Mach

Poliak

Matušková

et al. 2013

Genome Biology and Evolution

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Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial-targeting sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. As only α-MPP was duplicated in Neurospora, its single β–MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins showed that trypanosomal N-MTSs were comparable with N-MTSs from other organisms. N-MTS cleavage is mediated by a standard heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential for the parasite. Distinct Genes encode cp1 and cp2, and in the bloodstream forms the expression of cp1 is downregulated along with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, whereas cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not appear to have an ancient character proposed earlier for this eukaryote.

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Heterosubunit Composition and Crystal Structures of a Novel Bacterial M16B Metallopeptidase

Cited by 18 publications

References 46 publications

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

Conformational states and recognition of amyloidogenic peptides of human insulin-degrading enzyme

A Computational Study of the Glycine-Rich Loop of Mitochondrial Processing Peptidase

An Advanced System of the Mitochondrial Processing Peptidase and Core Protein Family in Trypanosoma brucei and Multiple Origins of the Core I Subunit in Eukaryotes

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