scholarly journals Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases

IUCrJ ◽  
2020 ◽  
Vol 7 (1) ◽  
pp. 18-29 ◽  
Author(s):  
Tibisay Guevara ◽  
Arturo Rodriguez-Banqueri ◽  
Miroslaw Ksiazek ◽  
Jan Potempa ◽  
F. Xavier Gomis-Rüth
Keyword(s):  
Catalytic Domain ◽  
Front Surface ◽  
Zinc Ion ◽  
Methanosarcina Acetivorans ◽  
Active Site Cleft ◽  
Switch Mechanism ◽  
Mature Enzyme ◽  
Catalytic Zinc ◽  
Matrix Metallopeptidases ◽  
Cysteine Switch

Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54–Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Å revealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1′. Moreover, residue Cys23 within a conserved cysteine–glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Å structure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1′ to S6′. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungusAspergillus fumigatus

2013 ◽  
Vol 69 (10) ◽  
pp. 1946-1957 ◽  
Author(s):  
Daniel Fernández ◽  
Silvia Russi ◽  
Josep Vendrell ◽  
Michel Monod ◽  
Irantzu Pallarès
Keyword(s):  
Catalytic Domain ◽  
Sequence Similarity ◽  
Pathogenic Fungi ◽  
Disulfide Bridge ◽  
Zinc Ion ◽  
Binary Complex ◽  
Active Site Cleft ◽  
Charged Amino Acid Residue ◽  
Polypeptide Chains ◽  
First Time

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family,Aspergillus fumigatusmetalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural–functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties ofA. fumigatusfungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

2005 ◽  
Vol 288 (6) ◽  
pp. L1132-L1138 ◽  
Author(s):  
Caitriona A. Buckley ◽  
Farshid N. Rouhani ◽  
Maryann Kaler ◽  
Barbara Adamik ◽  
Feras I. Hawari ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inhibitory Activity ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Mucin Production ◽  
Inactive State ◽  
Amino Terminal ◽  
Switch Mechanism ◽  
Cysteine Switch

TNF-α-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18–54 (N-TACE18–54) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE18–54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE18–54 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30–48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals The crystal structure of a 250-kDa heterotetrameric particle explains inhibition of sheddase meprin β by endogenous fetuin-B

2021 ◽  
Vol 118 (14) ◽  
pp. e2023839118
Author(s):  
Ulrich Eckhard ◽  
Hagen Körschgen ◽  
Nele von Wiegen ◽  
Walter Stöcker ◽  
F. Xavier Gomis-Rüth
Keyword(s):  
Crystal Structure ◽  
Glycan Structure ◽  
Type I ◽  
Zinc Ions ◽  
Transmembrane Segment ◽  
Protein Inhibitor ◽  
Catalytic Domains ◽  
Endogenous Protein ◽  
Switch Mechanism ◽  
Catalytic Zinc

Meprin β (Mβ) is a multidomain type-I membrane metallopeptidase that sheds membrane-anchored substrates, releasing their soluble forms. Fetuin-B (FB) is its only known endogenous protein inhibitor. Herein, we analyzed the interaction between the ectodomain of Mβ (MβΔC) and FB, which stabilizes the enzyme and inhibits it with subnanomolar affinity. The MβΔC:FB crystal structure reveals a ∼250-kDa, ∼160-Å polyglycosylated heterotetrameric particle with a remarkable glycan structure. Two FB moieties insert like wedges through a “CPDCP trunk” and two hairpins into the respective peptidase catalytic domains, blocking the catalytic zinc ions through an “aspartate switch” mechanism. Uniquely, the active site clefts are obstructed from subsites S4 to S10′, but S1 and S1′ are spared, which prevents cleavage. Modeling of full-length Mβ reveals an EGF-like domain between MβΔC and the transmembrane segment that likely serves as a hinge to transit between membrane-distal and membrane-proximal conformations for inhibition and catalysis, respectively.

Effects of reactive metabolites of oxygen and nitrogen on gelatinase A activity

1997 ◽  
Vol 273 (2) ◽  
pp. L445-L450 ◽  
Author(s):  
M. W. Owens ◽  
S. A. Milligan ◽  
D. Jourd'heuil ◽  
M. B. Grisham
Keyword(s):  
Nitric Oxide ◽  
Matrix Protein ◽  
Gelatinase A ◽  
Simultaneous Generation ◽  
Zinc Thiolate ◽  
Switch Mechanism ◽  
Increased Activity ◽  
Metalloproteinase Activity ◽  
Cysteine Switch ◽  
Proper Balance

The regulation of matrix metalloproteinase activity is crucial for maintaining the proper balance of tissue remodeling vs. injury. Metalloproteinase proenzymes are activated when the active site zinc is exposed via a cysteine switch mechanism. Peroxynitrite, the product generated from the interaction between nitric oxide and superoxide, has been shown to release zinc from zinc-thiolate groups, suggesting that it might alter metalloproteinase activity. This study examined the effects of nitric oxide and superoxide generators on gelatinase A activity. Results showed that nitric oxide alone had no effect on gelatinase A activity relative to control, whereas superoxide-derived metabolites increased activity. The simultaneous generation of both nitric oxide and superoxide caused an inhibition of gelatinase A activity. This inhibition was reversed by the addition of hemoglobin, superoxide dismutase, or sodium urate, suggesting that peroxynitrite and/or peroxynitrous acid caused the inhibition. Authentic peroxynitrite also inhibited gelatinase A activity. We postulate that the relative fluxes of nitric oxide and superoxide at sites of inflammation may modulate metalloproteinase activity and thus affect matrix protein metabolism.

scholarly journals Probing the Role of Divalent Metal Ions in a Bacterial Psychrophilic Metalloprotease: Binding Studies of an Enzyme in the Crystalline State by X-Ray Crystallography

2003 ◽  
Vol 185 (14) ◽  
pp. 4195-4203 ◽  
Author(s):  
Stephanie Ravaud ◽  
Patrice Gouet ◽  
Richard Haser ◽  
Nushin Aghajari
Keyword(s):  
Metal Ions ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Crystalline State ◽  
Zinc Ion ◽  
Binding Studies ◽  
X Ray ◽  
X Ray Crystallography ◽  
Catalytic Zinc

ABSTRACT The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated in Antarctica belongs to the clan of metzincins, for which a zinc ion is essential for catalytic activity. Binding studies in the crystalline state have been performed by X-ray crystallography in order to improve the understanding of the role of the zinc and calcium ions bound to this protease. Cocrystallization and soaking experiments with EDTA in a concentration range from 1 to 85 mM have resulted in five three-dimensional structures with a distinct number of metal ions occupying the ion-binding sites. Evolution of the structural changes observed in the vicinity of each cation-binding site has been studied as a function of the concentration of EDTA, as well as of time, in the presence of the chelator. Among others, we have found that the catalytic zinc ion was the first ion to be chelated, ahead of a weakly bound calcium ion (Ca 700) exclusive to the psychrophilic enzyme. Upon removal of the catalytic zinc ion, the side chains of the active-site residues His-173, His-179 and Tyr-209 shifted ∼4, 1.0, and 1.6 Å, respectively. Our studies confirm and also explain the sensitivity of PAP toward moderate EDTA concentrations and propose distinct roles for the calcium ions. A new crystal form of native PAP validates our previous predictions regarding the adaptation of this enzyme to cold environments as well as the proteolytic domain calcium ion being exclusive for PAP independent of crystallization conditions.

scholarly journals Myroilysin Is a New Bacterial Member of the M12A Family of Metzincin Metallopeptidases and Is Activated by a Cysteine Switch Mechanism

2017 ◽  
Vol 292 (13) ◽  
pp. 5195-5206 ◽  
Author(s):  
Dongqing Xu ◽  
Jiale Zhou ◽  
Xiangdi Lou ◽  
Jianhua He ◽  
Tingting Ran ◽  
...  
Keyword(s):  
Switch Mechanism ◽  
Cysteine Switch

Quantum Chemistry Study on the Interaction of the Exogenous Ligands and the Catalytic Zinc Ion in Matrix Metalloproteinases

2002 ◽  
Vol 106 (17) ◽  
pp. 4552-4559 ◽  
Author(s):  
Feng Cheng ◽  
Ruihao Zhang ◽  
Xiaomin Luo ◽  
Jianhua Shen ◽  
Xin Li ◽  
...  
Keyword(s):  
Matrix Metalloproteinases ◽  
Quantum Chemistry ◽  
Zinc Ion ◽  
Exogenous Ligands ◽  
Catalytic Zinc

scholarly journals X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides

2003 ◽  
Vol 185 (14) ◽  
pp. 4127-4135 ◽  
Author(s):  
David Mandelman ◽  
Anne Belaich ◽  
J. P. Belaich ◽  
Nushin Aghajari ◽  
Hugues Driguez ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Domain ◽  
Cellulose Degradation ◽  
Cellulase Activity ◽  
Structural Basis ◽  
Crystalline Cellulose ◽  
X Ray ◽  
Clostridium Cellulolyticum ◽  
Active Site Cleft ◽  
Cellulose Binding

ABSTRACT Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the β-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family IIIc cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.

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