Crystal structure and activity-based labeling reveal the mechanisms for linkage-specific substrate recognition by deubiquitinase USP9X

USP9X is a conserved deubiquitinase (DUB) that regulates multiple cellular processes. Dysregulation of USP9X has been linked to cancers and X-linked intellectual disability. Here, we report the crystal structure of the USP9X catalytic domain at 2.5-Å resolution. The structure reveals a canonical USP-fold comprised of fingers, palm, and thumb subdomains, as well as an unusual β-hairpin insertion. The catalytic triad of USP9X is aligned in an active configuration. USP9X is exclusively active against ubiquitin (Ub) but not Ub-like modifiers. Cleavage assays with di-, tri-, and tetraUb chains show that the USP9X catalytic domain has a clear preference for K11-, followed by K63-, K48-, and K6-linked polyUb chains. Using a set of activity-based diUb and triUb probes (ABPs), we demonstrate that the USP9X catalytic domain has an exo-cleavage preference for K48- and endo-cleavage preference for K11-linked polyUb chains. The structure model and biochemical data suggest that the USP9X catalytic domain harbors three Ub binding sites, and a zinc finger in the fingers subdomain and the β-hairpin insertion both play important roles in polyUb chain processing and linkage specificity. Furthermore, unexpected labeling of a secondary, noncatalytic cysteine located on a blocking loop adjacent to the catalytic site by K11-diUb ABP implicates a previously unreported mechanism of polyUb chain recognition. The structural features of USP9X revealed in our study are critical for understanding its DUB activity. The new Ub-based ABPs form a set of valuable tools to understand polyUb chain processing by the cysteine protease class of DUBs.

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001213 ◽

2018 ◽

Vol 293 (21) ◽

pp. 7993-8008 ◽

Cited By ~ 8

Author(s):

Subrata Debnath ◽

Dalibor Kosek ◽

Harichandra D. Tagad ◽

Stewart R. Durell ◽

Daniel H. Appella ◽

...

Keyword(s):

Crystal Structure ◽

Metal Ions ◽

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Phosphatases ◽

Random Order ◽

Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

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Structural and Functional Characteristics of ADAMTS-4 and ADAMTS-5: A Review

10.15688/nsr.jvolsu.2020.1.2 ◽

2020 ◽

pp. 12-21

Author(s):

Anastasia Korchagina ◽

Lyudmila Derevshchikova

Keyword(s):

Enzymatic Activity ◽

Binding Sites ◽

Catalytic Domain ◽

Structural Features ◽

Functional Characteristics ◽

Rich Domain ◽

Common Diseases ◽

The Moment ◽

Cysteine Rich Domain

ADAMTS-4 and -5 are aggrecanases that are involved in the development of osteoarthrosis, one of the most common diseases at the moment, due to which a large number of people suffer annually. By some estimates, 9.6% of men and 18% of women over the age of 60 have symptomatic osteoarthrosis. This review discusses the currently known data on the structural features and enzymatic activity of these enzymes. The structures of ADAMTS-4 and -5 are similar, they contain 7 domain sites: the signal section, the N-terminal prodomain, the catalytic domain, the disintegrin-like domain, the thrombospodin motif, the cysteine-rich domain, and the spacer domain. In addition to all these elements, ADAMTS-5 has an additional thrombospodin motif at the end. ADAMTS-4 and -5 cleaves aggrecan in 5 different binding sites. However, the Glu373-Ala374 site probably plays the most important role in the pathogenesis, since when this bond is broken, the whole aggrecan molecule loses its integrity, which leads to the destruction of cartilage and the development of the disease. In the course of the analysis of the information, the authors have found that the participation of ADAMTS-4 and -5 in the development of osteoarthritis varies greatly depending on the type of organism. The researchers have established that in mice ADAMTS-4 plays the largest role in the destruction of aggrecan, while in humans ADAMTS-5 or both aggrecanases influence the development of osteoarthritis. The revealed differences are not fully described; therefore, this review summarizes the already known results in this area, which will facilitate further research in this direction.

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The Crystal Structure of a Streptomyces thermoviolaceus Thermophilic Chitinase Known for Its Refolding Efficiency

International Journal of Molecular Sciences ◽

10.3390/ijms21082892 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2892

Author(s):

Piotr H. Malecki ◽

Magdalena Bejger ◽

Wojciech Rypniewski ◽

Constantinos E. Vorgias

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Structural Features ◽

Salt Bridges ◽

Aromatic Rings ◽

Tim Barrel ◽

Trimmed Surface ◽

Binding Cleft ◽

Surface Loops ◽

Structure Of Proteins

Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, because chitin, while being the world’s second most abundant biopolymer, is also a recalcitrant substrate. The crystal structure of a thermostable chitinase from Streptomyces thermoviolaceus (StChi40) has been solved revealing a β/α-barrel (TIM-barrel) fold with an α+β insertion domain. This is the first chitinase structure of the multi-chitinase system of S. thermoviolaceus. The protein is also known to refold efficiently after thermal or chemical denaturation. StChi40 is structurally close to the catalytic domain of psychrophilic chitinase B from Arthrobacter TAD20. Differences are noted in comparison to the previously examined chitinases, particularly in the substrate-binding cleft. A comparison of the thermophilic enzyme with its psychrophilic homologue revealed structural features that could be attributed to StChi40’s thermal stability: compactness of the structure with trimmed surface loops and unique disulfide bridges, one of which is additionally stabilized by S–π interactions with aromatic rings. Uncharacteristically for thermophilic proteins, StChi40 has fewer salt bridges than its mesophilic and psychrophilic homologues.

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Homology modelling and structural analysis of human arylamine N-acetyltransferase NAT1: evidence for the conservation of a cysteine protease catalytic domain and an active-site loop

Biochemical Journal ◽

10.1042/bj3560327 ◽

2001 ◽

Vol 356 (2) ◽

pp. 327-334 ◽

Cited By ~ 20

Author(s):

Fernando RODRIGUES-LIMA ◽

Claudine DELOMÉNIE ◽

Geoffrey H. GOODFELLOW ◽

Denis M. GRANT ◽

Jean-Marie DUPRET

Keyword(s):

Crystal Structure ◽

Cysteine Protease ◽

Active Site ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Homology Modelling ◽

Metabolic Activation ◽

Cysteine Proteases ◽

Catalytic Triad ◽

Quality Model

Arylamine N-acetyltransferases (EC 2.3.1.5) (NATs) catalyse the biotransformation of many primary arylamines, hydrazines and their N-hydroxylated metabolites, thereby playing an important role in both the detoxification and metabolic activation of numerous xenobiotics. The recently published crystal structure of the Salmonella typhimurium NAT (StNAT) revealed the existence of a cysteine protease-like (Cys-His-Asp) catalytic triad. In the present study, a three-dimensional homology model of human NAT1, based upon the crystal structure of StNAT [Sinclair, Sandy, Delgoda, Sim and Noble (2000) Nat. Struct. Biol. 7, 560–564], is demonstrated. Alignment of StNAT and NAT1, together with secondary structure predictions, have defined a consensus region (residues 29–131) in which 37% of the residues are conserved. Homology modelling provided a good quality model of the corresponding region in human NAT1. The location of the catalytic triad was found to be identical in StNAT and NAT1. Comparison of active-site structural elements revealed that a similar length loop is conserved in both species (residues 122–131 in NAT1 model and residues 122–133 in StNAT). This observation may explain the involvement of residues 125, 127 and 129 in human NAT substrate selectivity. Our model, and the fact that cysteine protease inhibitors do not affect the activity of NAT1, suggests that human NATs may have adapted a common catalytic mechanism from cysteine proteases to accommodate it for acetyl-transfer reactions.

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Crystal structure of GH30-7 endoxylanase C from the filamentous fungus Talaromyces cellulolyticus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20009024 ◽

2020 ◽

Vol 76 (8) ◽

pp. 341-349

Author(s):

Yusuke Nakamichi ◽

Tatsuya Fujii ◽

Masahiro Watanabe ◽

Akinori Matsushika ◽

Hiroyuki Inoue

Keyword(s):

Crystal Structure ◽

Glucuronic Acid ◽

Catalytic Domain ◽

Catalytic Efficiency ◽

Structural Features ◽

Hydroxyl Group ◽

Glycoside Hydrolase Family ◽

Talaromyces Cellulolyticus ◽

Cellulose Binding ◽

Hydrolase Family

GH30-7 endoxylanase C from the cellulolytic fungus Talaromyces cellulolyticus (TcXyn30C) belongs to glycoside hydrolase family 30 subfamily 7, and specifically releases 22-(4-O-methyl-α-D-glucuronosyl)-xylobiose from glucuronoxylan, as well as various arabino-xylooligosaccharides from arabinoxylan. TcXyn30C has a modular structure consisting of a catalytic domain and a C-terminal cellulose-binding module 1 (CBM1). In this study, the crystal structure of a TcXyn30C mutant which lacks the CBM1 domain was determined at 1.65 Å resolution. The structure of the active site of TcXyn30C was compared with that of the bifunctional GH30-7 xylanase B from T. cellulolyticus (TcXyn30B), which exhibits glucuronoxylanase and xylobiohydrolase activities. The results revealed that TcXyn30C has a conserved structural feature for recognizing the 4-O-methyl-α-D-glucuronic acid (MeGlcA) substituent in subsite −2b. Additionally, the results demonstrated that Phe47 contributes significantly to catalysis by TcXyn30C. Phe47 is located in subsite −2b and also near the C-3 hydroxyl group of a xylose residue in subsite −2a. Substitution of Phe47 with an arginine residue caused a remarkable decrease in the catalytic efficiency towards arabinoxylan, suggesting the importance of Phe47 in arabinoxylan hydrolysis. These findings indicate that subsite −2b of TcXyn30C has unique structural features that interact with arabinofuranose and MeGlcA substituents.

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Structural similarities of Na,K-ATPase and SERCA, the Ca2+-ATPase of the sarcoplasmic reticulum

Biochemical Journal ◽

10.1042/bj3560685 ◽

2001 ◽

Vol 356 (3) ◽

pp. 685-704 ◽

Cited By ~ 78

Author(s):

Kathleen J. SWEADNER ◽

Claudia DONNET

Keyword(s):

Crystal Structure ◽

Sarcoplasmic Reticulum ◽

Binding Sites ◽

Atp Hydrolysis ◽

Structural Features ◽

Cleavage Sites ◽

Transmembrane Helices ◽

Transmembrane Topology ◽

Key Features ◽

P Type

The crystal structure of SERCA1a (skeletal-muscle sarcoplasmic-reticulum/endoplasmic-reticulum Ca2+-ATPase) has recently been determined at 2.6 Å (note 1 Å = 0.1nm) resolution [Toyoshima, Nakasako, Nomura and Ogawa (2000) Nature (London) 405, 647–655]. Other P-type ATPases are thought to share key features of the ATP hydrolysis site and a central core of transmembrane helices. Outside of these most-conserved segments, structural similarities are less certain, and predicted transmembrane topology differs between subclasses. In the present review the homologous regions of several representative P-type ATPases are aligned with the SERCA sequence and mapped on to the SERCA structure for comparison. Homology between SERCA and the Na,K-ATPase is more extensive than with any other ATPase, even PMCA, the Ca2+-ATPase of plasma membrane. Structural features of the Na,K-ATPase are projected on to the Ca2+-ATPase crystal structure to assess the likelihood that they share the same fold. Homology extends through all ten transmembrane spans, and most insertions and deletions are predicted to be at the surface. The locations of specific residues are examined, such as proteolytic cleavage sites, intramolecular cross-linking sites, and the binding sites of certain other proteins. On the whole, the similarity supports a shared fold, with some particular exceptions.

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Crystal structure of the substrate-recognition domain of theShigellaE3 ligase IpaH9.8

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16002715 ◽

2016 ◽

Vol 72 (4) ◽

pp. 269-275 ◽

Cited By ~ 5

Author(s):

Kenji Takagi ◽

Minsoo Kim ◽

Chihiro Sasakawa ◽

Tsunehiro Mizushima

Keyword(s):

Crystal Structure ◽

Pathogenic Bacteria ◽

Inflammatory Responses ◽

Substrate Recognition ◽

Structural Features ◽

Host Cells ◽

Structural Basis ◽

Global Public Health ◽

Leucine Rich Repeats ◽

Lrr Domain

Infectious diseases caused by bacteria have significant impacts on global public health. During infection, pathogenic bacteria deliver a variety of virulence factors, called effectors, into host cells. TheShigellaeffector IpaH9.8 functions as an ubiquitin ligase, ubiquitinating the NF-κB essential modulator (NEMO)/IKK-γ to inhibit host inflammatory responses. IpaH9.8 contains leucine-rich repeats (LRRs) involved in substrate recognition and an E3 ligase domain. To elucidate the structural basis of the function of IpaH9.8, the crystal structure of the LRR domain ofShigellaIpaH9.8 was determined and this structure was compared with the known structures of other IpaH family members. This model provides insights into the structural features involved in substrate specificity.

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Structure of the catalytic domain of theSalmonellavirulence factor SseI

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444912039042 ◽

2012 ◽

Vol 68 (12) ◽

pp. 1613-1621 ◽

Cited By ~ 13

Author(s):

Shyam S. Bhaskaran ◽

C. Erec Stebbins

Keyword(s):

Crystal Structure ◽

Virulence Factor ◽

Cysteine Protease ◽

Catalytic Domain ◽

Bacterial Pathogens ◽

Catalytic Triad ◽

Host Cells ◽

Eukaryotic Cells ◽

Acyltransferase Activity ◽

Systemic Infections

SseI is secreted into host cells bySalmonellaand contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.

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