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Conformational analysis of isolated domains of Helicobacter pylori CagA.

Woon AP, Tohidpour A, Alonso H, Saijo-Hamano Y, Kwok T, Roujeinikova A - PLoS ONE (2013)

Bottom Line: All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners.Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways.Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

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Related in: MedlinePlus

CD spectra of the recombinant CagA fragments.Ellipticity in the far-UV range (200–260 nm) is plotted for (a) CagA-N at 0.075 mg/ml, (b) CagA-M at 0.05 mg/ml, (c) CagA-Mc at 0.1 mg/ml and (d) Cag-R at 0.15 mg/ml.
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pone-0079367-g003: CD spectra of the recombinant CagA fragments.Ellipticity in the far-UV range (200–260 nm) is plotted for (a) CagA-N at 0.075 mg/ml, (b) CagA-M at 0.05 mg/ml, (c) CagA-Mc at 0.1 mg/ml and (d) Cag-R at 0.15 mg/ml.

Mentions: The secondary structure of CagA-N, CagA-M, CagA-Mc and CagA-R was investigated using CD. The CD spectra of CagA-N, CagA-M and CagA-Mc (Figure 3a,b and c) exhibited double minima at approximately 208 and 222 nm, profiles which are characteristic of proteins with high α-helical content. Estimation of the α-helix and β-sheet content in the secondary structure using K2d [28] gave values that are close to those derived from the crystal structure of the truncated CagA variant [19,20] or predicted from sequence analysis using Jpred3 [29] (Table 1), indicating that the purified fragments CagA-N, CagA-M and CagA-Mc were folded. In contrast, the far-UV CD spectrum of CagA-R showed a single sharp minimum at around 203 nm (Figure 3d) and a relatively low ellipticity above 210 nm, indicative of poorly structured conformations with low content of secondary structure, a feature that is often attributed to intrinsically disordered proteins of premolten globule (PMG) type [30-33]. Analysis of the CD spectrum of CagA-R indicated 55% random coil and 6% helical content (the remainder was calculated to be β-sheet, although it is difficult to accurately quantify β-sheet content by CD).


Conformational analysis of isolated domains of Helicobacter pylori CagA.

Woon AP, Tohidpour A, Alonso H, Saijo-Hamano Y, Kwok T, Roujeinikova A - PLoS ONE (2013)

CD spectra of the recombinant CagA fragments.Ellipticity in the far-UV range (200–260 nm) is plotted for (a) CagA-N at 0.075 mg/ml, (b) CagA-M at 0.05 mg/ml, (c) CagA-Mc at 0.1 mg/ml and (d) Cag-R at 0.15 mg/ml.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815135&req=5

pone-0079367-g003: CD spectra of the recombinant CagA fragments.Ellipticity in the far-UV range (200–260 nm) is plotted for (a) CagA-N at 0.075 mg/ml, (b) CagA-M at 0.05 mg/ml, (c) CagA-Mc at 0.1 mg/ml and (d) Cag-R at 0.15 mg/ml.
Mentions: The secondary structure of CagA-N, CagA-M, CagA-Mc and CagA-R was investigated using CD. The CD spectra of CagA-N, CagA-M and CagA-Mc (Figure 3a,b and c) exhibited double minima at approximately 208 and 222 nm, profiles which are characteristic of proteins with high α-helical content. Estimation of the α-helix and β-sheet content in the secondary structure using K2d [28] gave values that are close to those derived from the crystal structure of the truncated CagA variant [19,20] or predicted from sequence analysis using Jpred3 [29] (Table 1), indicating that the purified fragments CagA-N, CagA-M and CagA-Mc were folded. In contrast, the far-UV CD spectrum of CagA-R showed a single sharp minimum at around 203 nm (Figure 3d) and a relatively low ellipticity above 210 nm, indicative of poorly structured conformations with low content of secondary structure, a feature that is often attributed to intrinsically disordered proteins of premolten globule (PMG) type [30-33]. Analysis of the CD spectrum of CagA-R indicated 55% random coil and 6% helical content (the remainder was calculated to be β-sheet, although it is difficult to accurately quantify β-sheet content by CD).

Bottom Line: All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners.Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways.Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

ABSTRACT
The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions.

Show MeSH
Related in: MedlinePlus