Crucial Role of Perfringolysin O D1 Domain in Orchestrating Structural Transitions Leading to Membrane-perforating Pores

Kacprzyk-Stokowiec, Aleksandra; Kulma, Magdalena; Traczyk, Gabriela; Kwiatkowska, Katarzyna; Sobota, Andrzej; Dadlez, Michał

doi:10.1074/jbc.m114.577981

Cited by 16 publications

(12 citation statements)

References 43 publications

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“…The method was performed as described previously [37], with minor modifications. Initially, the list of peptides was established by diluting each analyzed protein to 5–10 μM in a non-deuterated buffer (20 mM Hepes pH 6, 100mM KCl).…”

Section: Methodsmentioning

confidence: 99%

Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study

Skrajna

Yang

Tarnowski

et al. 2016

Journal of Molecular Biology

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Histone pre-mRNAs are cleaved at the 3′ end by a complex that contains U7 snRNP, the FLICE-Associated Huge protein (FLASH) and Histone pre-mRNA Cleavage Complex (HCC) consisting of several polyadenylation factors. Within the complex, the N-terminus of FLASH interacts with the N-terminus of the U7 snRNP protein Lsm11 and together they recruit the HCC. FLASH through its distant C-terminus independently interacts with the C-terminal SANT/Myb-like domain of Nuclear Protein, Ataxia-Telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S-phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium (H/D) exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both H/D exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.

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Section: Methodsmentioning

confidence: 99%

Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study

Skrajna

Yang

Tarnowski

et al. 2016

Journal of Molecular Biology

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“…Two control experiments were performed to account for in-and out-exchange artifacts, as described previously (Kacprzyk-Stokowiec et al, 2014). In brief, to assess minimum exchange (in-exchange control), D 2 O reaction buffer was added to stop buffer that had been cooled on ice prior to addition of protein stock, and this mixture was immediately subjected to pepsin digestion and LC-MS analysis as described above.…”

Section: Hydrogen-deuterium Exchangementioning

confidence: 99%

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen–deuterium exchange

Premchandar

Kupniewska

Tarnowski

et al. 2015

Journal of Structural Biology

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Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.

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“…D3 does not directly contact D4, but forms an interface with both D1 and D2 that connects D3 with D4 ( Figure 1 , Figure 5 b) [ 30 , 31 , 43 ]. The stability of D1 increases upon membrane binding, which may be important for disengagement of the D2,3 interface [ 71 ]. When D1 is destabilized by the substitution of a tryptophan residue (Trp-165 in PFO), the interface between D2,3 fails to disengage [ 63 , 71 ].…”

Section: Molecular Mechanism Of Pore Formationmentioning

confidence: 99%

“…The stability of D1 increases upon membrane binding, which may be important for disengagement of the D2,3 interface [ 71 ]. When D1 is destabilized by the substitution of a tryptophan residue (Trp-165 in PFO), the interface between D2,3 fails to disengage [ 63 , 71 ]. Additionally, Wade et al [ 72 ] recently showed that the rotation of β5 away from the core β-sheet in D3 allows the formation of a strong intermolecular electrostatic interaction, which drives the transition from the prepore to the pore by providing the free energy necessary to disrupt the interface between D3 and D1,2.…”

Section: Molecular Mechanism Of Pore Formationmentioning

confidence: 99%

“…A quorum-sensing system homologous to the accessory gene regulator system ( agr ) of Staphylococcus aureus was found in C. perfringens , which could regulate the expression of several virulence factors in response to external stimuli [ 74 , 75 , 76 ]. Ohtani et al [ 71 ] and Vidal et al [ 76 ] showed that the expression of PFO and α toxin are regulated by the agr system, as the mutation of the agr locus influenced the early log-phase expression of both toxins. C. perfringens also harbors an additional quorum-sensing system homologous to the luxS system [ 77 ].…”

Section: Genetic Regulationmentioning

confidence: 99%

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Perfringolysin O: The Underrated Clostridium perfringens Toxin?

Verherstraeten

Goossens

Valgaeren

et al. 2015

Toxins

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The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.

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Crucial Role of Perfringolysin O D1 Domain in Orchestrating Structural Transitions Leading to Membrane-perforating Pores

Cited by 16 publications

References 43 publications

Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study

Mapping the Interaction Network of Key Proteins Involved in Histone mRNA Generation: A Hydrogen/Deuterium Exchange Study

Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen–deuterium exchange

Perfringolysin O: The Underrated Clostridium perfringens Toxin?

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