Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

Toda, Ayako; Tsurumura, Toshiharu; Yoshida, Toru; Tsumori, Yayoi; Tsuge, Hideaki

doi:10.1074/jbc.m115.653220

Cited by 25 publications

(28 citation statements)

References 55 publications

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“…Similarly to TcdB and LT, IbpA also recognizes Rho-family GTPases by binding to the Switch II region 60 . Moreover, C3 exoenzyme from C. botulinum , which specifically modifies RhoA/B/C but not other members of either Rho or Ras subfamilies, recognizes Switch II and interswitch regions in addition to Switch I 61 .…”

Section: Discussionmentioning

confidence: 99%

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Biancucci

Rabideau

et al. 2017

Biochemistry

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Ras/Rap1-specific endopeptidase (RRSP) is a cytotoxic effector domain of Multifunctional-autoprocessing repeats-in-toxins (MARTX) toxin of highly virulent strains of Vibrio vulnificus. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1 within the Switch I region between Y32 and D33. Although the RRSP processing site is highly conserved among small GTPases, only Ras and Rap1 have been identified as proteolytic substrates. Here we report that Y32-D33 residues at the scissile bond play an important role for RRSP substrate recognition, while nucleotide state of Ras has only a minimal effect. In addition, substrate specificity is generated by residues across the entire Switch I region. Indeed, swapping the Ras Switch I region into either RalA or RhoA, GTPases that are not recognized by RRSP, generated chimeras that are substrates of RRSP. However, a difference in processing efficiency of the Ras Switch I in the context of Ras, RalA, or RhoA indicates that protein regions outside the Ras Switch I also contribute to efficient RRSP substrate recognition. Moreover, we show that synthetic peptides corresponding to the Ras and Rap1, but not RalA, Switch I regions are cleaved by RRSP demonstrating sequence specific substrate recognition. In conclusion, this work demonstrates that the GTPase recognition of RRSP is nucleotide independent, and is mainly driven by the Ras and Rap1 Switch I loop and also influenced by additional protein-protein interactions, increasing the substrate specificity of RRSP.

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Section: Discussionmentioning

confidence: 99%

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Biancucci

Rabideau

et al. 2017

Biochemistry

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“…1b) (26). Though it has not been confirmed until recently, we directly observed substrate recognition via the ARTT-loop in C3-RhoA complex structure (25). Within five loops of the C3 exoenzyme, the ARTT-loop determines the acceptor amino acid: Gln183 (QXE) on the second turn of ARTT-loop recognizes Asn41 of RhoA.…”

Section: Adp-ribosylationmentioning

confidence: 91%

“…Furthermore Tyr180 on the first turn of the ARTT-loop is also important to recognize the hydrophobic patch of RhoA comprising Val43, Ala56, and Trp58. This interaction facilitates ADP-ribosylation of the specific asparagine of RhoA (25,27). However, the target residue recognition mechanism of ARTs via the ARTT-loop is still controversial because of the lack of substrate complex structures.…”

Section: Adp-ribosylationmentioning

confidence: 99%

“…Only three complex structures of ART toxin with its substrate are available (23)(24)(25). Among these, it is well known how C3 exoenzyme recognizes its specific substrate protein (RhoA) and acceptor amino acid via the ARTT-loop.…”

Section: Adp-ribosylationmentioning

confidence: 99%

“…Eukaryotic enzyme poly-ADP-ribose polymerases (PARPs) can mono-ADP-ribosylate double-stranded DNA ends, which can be reversed by several known . The present study is the first report of the detailed substrate recognition and ADP-ribosylation mechanisms in the pierisin family DNA-targeting ARTs (22).Only three complex structures of ART toxin with its substrate are available (23)(24)(25). Among these, it is well known how C3 exoenzyme recognizes its specific substrate protein (RhoA) and acceptor amino acid via the ARTT-loop.…”

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confidence: 99%

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Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP

Yoshida

Tsuge

2018

Journal of Biological Chemistry

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ScARP from the bacterium Streptomyces coelicolor belongs to the pierisin family of DNA-targeting ADP-ribosyltransferases (ARTs). These enzymes ADP-ribosylate the N 2 amino groups of guanine residues in DNA to yield N 2 -(ADP-ribos-1-yl)-2'-deoxyguanosine. Although the structures of pierisin-1 and Scabin were revealed recently, the substrate recognition mechanisms remain poorly understood because of the lack of a substrate-binding structure. Here, we report the apo structure of ScARP and of ScARP bound to NADH and its GDP substrate at 1.50 and 1.57 Å resolutions, respectively. The bound structure revealed that the guanine of GDP is trapped between N-ribose of NADH and Trp159. Interestingly, N 2 and N 3 of guanine formed hydrogen bonds with the OE1 and NE2 atoms of Gln162, respectively. We directly observed that the ADP-ribosylating toxin turn-turn (ARTT)-loop including Trp159 and Gln162 plays a key role in the specificity of DNA-targeting guanine-specific ART as well as protein-targeting ARTs such as C3 exoenzyme. We propose that the ARTT-loop recognition is a common substrate recognition mechanism in the pierisin family. Furthermore, this complex structure sheds light on similarities and differences among two subclasses that are distinguished by conserved structural motifs: H-Y-E in the ARTD subfamily and R-S-E in the ARTC subfamily. The spatial arrangements of the electrophile and nucleophile were the same, providing the first evidence for a common reaction mechanism in these ARTs. ARTC (including ScARP) uses the ARTT-loop for substrate recognition, whereas ARTD (represented by Arr) uses the C-terminal helix instead of the ARTT-loop. These observations could help inform efforts to improve ART inhibitors. ADP-ribosylationis an important post-translational modification observed in all living organisms. Many bacterial mono-ADP-ribosyltransferases (ARTs) are known to attach the ADP-ribosyl moiety to specific target proteins and residues (1,2). The cholera toxin ADP-ribosylates arginine residues in G proteins (3), whereas the pertussis toxin ADP-ribosylates a cysteine residue (4). Diphtheria toxin and Pseudomonas aeruginosa exotoxin ADP-ribosylate diphthamide, a modified histidine in elongation factor 2 (5,6). Furthermore, Clostridium botulinum C3 exoenzyme ADP-ribosylates Asn41 of RhoA (7,8) and Clostridium perfringens Iota toxin A subunit (Ia) ADP-ribosylates Arg177 of actin (9,10 ARTs fall into two major subclasses that are distinguished by conserved structural motifs: H-Y-E in the ARTD subfamily (related to Diphtheria toxin) and R-S-E in the ARTC subfamily (related to cholera toxin and clostridial toxins (C3 and Ia)). Traditionally, ADP-ribosylation has been considered a protein modification. However, emerging evidence suggests that DNA ADP-ribosylation is also common. The first DNA-targeting ART was found in pierid butterflies and was thus named pierisin (11,12). Pierisin ADP-ribosylates calf thymus DNA containing dG-dC and N 2 amino group of the guanine residue in DNA to yield N 2 -(ADP-ribos-1-yl)...

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Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs

Delgado-Buenrostro

Mújica

Chiquete-Félix

et al. 2016

Molecular Reproduction Devel

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Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

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Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

Cited by 25 publications

References 55 publications

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase

Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP

Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs

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