Structural and Functional Studies Indicate That Shigella VirA Is Not a Protease and Does Not Directly Destabilize Microtubules

Germane, Katherine L.; Ohi, Ryoma; Goldberg, Marcia B.; Spiller, Benjamin W.

doi:10.1021/bi801533k

Cited by 31 publications

(37 citation statements)

References 19 publications

(25 reference statements)

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“…The structure of an N-terminally truncated VirA, crystallized in a space group different from the full-length protein described here, appeared in press when this manuscript was under review (Germane et al 2008). The results presented here, in particular concerning the novel fold of VirA and the lack of detectable enzymatic activity, are fully consistent with their observations.…”

Section: Note Added In Proofsupporting

confidence: 83%

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

Davis¹,

Wang

Tropea³

et al. 2008

Protein Science

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VirA, a secreted effector protein from Shigella sp., has been shown to be necessary for its virulence. It was also reported that VirA might be related to papain-like cysteine proteases and cleave a-tubulin, thus facilitating intracellular spreading. We have now determined the crystal structure of VirA at 3.0 Å resolution. The shape of the molecule resembles the letter ''V,'' with the residues in the N-terminal third of the 45-kDa molecule (some of which are disordered) forming one clearly identifiable domain, and the remainder of the molecule completing the V-like structure. The fold of VirA is unique and does not resemble that of any known protein, including papain, although its N-terminal domain is topologically similar to cysteine protease inhibitors such as stefin B. Analysis of the sequence conservation between VirA and its Escherichia coli homologs EspG and EspG2 did not result in identification of any putative protease-like active site, leaving open a possibility that the biological function of VirA in Shigella virulence may not involve direct proteolytic activity.

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Section: Note Added In Proofsupporting

confidence: 83%

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

Davis¹,

Wang

Tropea³

et al. 2008

Protein Science

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“…MT Disassembly-MT disassembly was assayed essentially as described (12,36,38). Surfaces of double-stick tape flow cells were coated with 1 mg/ml biotinylated BSA followed by 0.1 mg/ml streptavidin.…”

Section: Purification Of His-tagged Proteins-gateway Expression Vectomentioning

confidence: 99%

“…CopN from C. pneumonia was particularly effective at inhibiting MT formation, showing near-complete inhibition at 25 M. Consistent with the observation that CopN binds free ␣␤-tubulin but not MTs, we also observed that it inhibited ␣␤-tubulin polymerization but did not promote depolymerization of MTs. We evaluated the ability of CopN to destabilize MTs by mixing CopN with preformed rhodamine-labeled MT as described (36,38). As shown in Fig.…”

Section: Domain Mapping Experiments Demonstrate That Copn Encodes Thrmentioning

confidence: 99%

The Chlamydia Effector Chlamydial Outer Protein N (CopN) Sequesters Tubulin and Prevents Microtubule Assembly

Archuleta

English

et al. 2011

Journal of Biological Chemistry

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Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system "plug" proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to ␣␤-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free ␣␤-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of ␣␤-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.Chlamydia species, the causative agents of a range of human diseases, including genital, ocular, and respiratory infection (3, 14 -16), are obligate intracellular pathogens with a biphasic life cycle consisting of metabolically inactive elementary bodies and metabolically active reticulate bodies (17). Infection involves attachment and cellular uptake of elementary bodies and subsequent differentiation into reticulate bodies. The reticulate bodies remain in endosome-derived membrane vacuoles termed "inclusions," which migrate to the microtubule-organizing center in a dynein-dependent process (18 -20). After

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“…The Shigella effector VirA also interacts with ␣-and ␤-tubulin heterodimers but induces microtubule destabilization through an ␣-tubulin-specific cysteine protease-like activity (51,52,53). Interestingly, VirA is not a protease, nor does it directly sever or destabilize microtubules (11,15).…”

Section: Vol 78 2010mentioning

confidence: 99%

EseG, an Effector of the Type III Secretion System of Edwardsiella tarda , Triggers Microtubule Destabilization

Xie

Zheng

et al. 2010

Infect Immun

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Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD 50 ) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 ␤-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. (EseG 142-192 ), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG

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Structural and Functional Studies Indicate That Shigella VirA Is Not a Protease and Does Not Directly Destabilize Microtubules

Cited by 31 publications

References 19 publications

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

The Chlamydia Effector Chlamydial Outer Protein N (CopN) Sequesters Tubulin and Prevents Microtubule Assembly

EseG, an Effector of the Type III Secretion System of Edwardsiella tarda , Triggers Microtubule Destabilization

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