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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

SEC and molecular weight (MW) and hydrodynamic radius determination of CagA-N (a), CagA-Mc (b) and CagA-R (c).Green dots superimposed on the peak indicate the MW as shown on the left-hand y-axis. Red dots represent the hydrodynamic radius calculated over the central portion of the elution peak (shown by UV trace in blue). The hydrodynamic radius values are shown on the right-hand y-axis.
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pone-0079367-g004: SEC and molecular weight (MW) and hydrodynamic radius determination of CagA-N (a), CagA-Mc (b) and CagA-R (c).Green dots superimposed on the peak indicate the MW as shown on the left-hand y-axis. Red dots represent the hydrodynamic radius calculated over the central portion of the elution peak (shown by UV trace in blue). The hydrodynamic radius values are shown on the right-hand y-axis.

Mentions: CagA-N eluted as a single, monodisperse peak with the derived MW value of 22.5±2.3 kDa (Figure 4a; we estimated the accuracy of the weight determination as approximately 10% from the quality of the BSA standard). This value is consistent with a monomer. The apparent hydrodynamic radius of the particles in this peak was 2.5±0.3 nm (Figure 4a), which is in excellent agreement with the value of the hydrodynamic radius calculated using the 3D coordinates of the N-terminal CagA domain (region 24-221) in the crystal [19] (2.5 nm).


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)

SEC and molecular weight (MW) and hydrodynamic radius determination of CagA-N (a), CagA-Mc (b) and CagA-R (c).Green dots superimposed on the peak indicate the MW as shown on the left-hand y-axis. Red dots represent the hydrodynamic radius calculated over the central portion of the elution peak (shown by UV trace in blue). The hydrodynamic radius values are shown on the right-hand y-axis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079367-g004: SEC and molecular weight (MW) and hydrodynamic radius determination of CagA-N (a), CagA-Mc (b) and CagA-R (c).Green dots superimposed on the peak indicate the MW as shown on the left-hand y-axis. Red dots represent the hydrodynamic radius calculated over the central portion of the elution peak (shown by UV trace in blue). The hydrodynamic radius values are shown on the right-hand y-axis.
Mentions: CagA-N eluted as a single, monodisperse peak with the derived MW value of 22.5±2.3 kDa (Figure 4a; we estimated the accuracy of the weight determination as approximately 10% from the quality of the BSA standard). This value is consistent with a monomer. The apparent hydrodynamic radius of the particles in this peak was 2.5±0.3 nm (Figure 4a), which is in excellent agreement with the value of the hydrodynamic radius calculated using the 3D coordinates of the N-terminal CagA domain (region 24-221) in the crystal [19] (2.5 nm).

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