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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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SDS-PAGE showing the time-course of digestion of CagA-M by trypsin.The arrow indicates a relatively stable core fragment CagA-Mc.
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pone-0079367-g002: SDS-PAGE showing the time-course of digestion of CagA-M by trypsin.The arrow indicates a relatively stable core fragment CagA-Mc.

Mentions: To investigate the boundaries of each domain's stable core resistant to proteolysis, we carried out digests with trypsin and monitored the time-course of degradation by SDS-PAGE. CagA-R was fully degraded to small peptides within 30 min of digestion (Figure S2 in File S1), indicating that all the potential trypsin cleavage sites are exposed and, therefore, CagA-R is either fully unfolded or intrinsically disordered. In contrast, the time-course of degradation of CagA-M (Figure 2) demonstrated accumulation of a relatively stable fragment (the core fragment termed hereafter CagA-Mc) before further degradation into shorter products. To determine its boundaries, the molecular mass of CagA-Mc was measured by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS), yielding the value of 60,300 ± 100 Da. N-terminal sequencing identified the first five amino acid residues of CagA-Mc as GNFSK. This indicated that the N-terminus of CagA-M remained intact and defined CagA-Mc as fragment 267-807 with excellent agreement between the experimental and the calculated (60,288 Da) mass values. This result suggested that region 808-880 of CagA-M is flexible and accessible to protease and that the remainder has a compact and stable fold, which is consistent with the previous crystallographic studies on CagA(1-876) [19] that showed residues 825-876 were structurally disordered. This result is also in line with the previous limited trypsin proteolysis study of recombinant CagA(1-876) [19], which reported cleavage at a close albeit distinct site 829. We then cloned, expressed and purified the stable core fragment CagA-Mc and included it into subsequent biophysical experiments. In the tryptic digestion of CagA-N (Figure S2 in File S1), the band corresponding to intact protein (25 kDa) was present even after 3 hrs, demonstrating that this fragment is more resistant to proteolysis than CagA-M and CagA-R. Together with the fact that no accumulation of metastable fragments was observed, this suggested that fragment CagA-N folds into a single stable domain.


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)

SDS-PAGE showing the time-course of digestion of CagA-M by trypsin.The arrow indicates a relatively stable core fragment CagA-Mc.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079367-g002: SDS-PAGE showing the time-course of digestion of CagA-M by trypsin.The arrow indicates a relatively stable core fragment CagA-Mc.
Mentions: To investigate the boundaries of each domain's stable core resistant to proteolysis, we carried out digests with trypsin and monitored the time-course of degradation by SDS-PAGE. CagA-R was fully degraded to small peptides within 30 min of digestion (Figure S2 in File S1), indicating that all the potential trypsin cleavage sites are exposed and, therefore, CagA-R is either fully unfolded or intrinsically disordered. In contrast, the time-course of degradation of CagA-M (Figure 2) demonstrated accumulation of a relatively stable fragment (the core fragment termed hereafter CagA-Mc) before further degradation into shorter products. To determine its boundaries, the molecular mass of CagA-Mc was measured by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS), yielding the value of 60,300 ± 100 Da. N-terminal sequencing identified the first five amino acid residues of CagA-Mc as GNFSK. This indicated that the N-terminus of CagA-M remained intact and defined CagA-Mc as fragment 267-807 with excellent agreement between the experimental and the calculated (60,288 Da) mass values. This result suggested that region 808-880 of CagA-M is flexible and accessible to protease and that the remainder has a compact and stable fold, which is consistent with the previous crystallographic studies on CagA(1-876) [19] that showed residues 825-876 were structurally disordered. This result is also in line with the previous limited trypsin proteolysis study of recombinant CagA(1-876) [19], which reported cleavage at a close albeit distinct site 829. We then cloned, expressed and purified the stable core fragment CagA-Mc and included it into subsequent biophysical experiments. In the tryptic digestion of CagA-N (Figure S2 in File S1), the band corresponding to intact protein (25 kDa) was present even after 3 hrs, demonstrating that this fragment is more resistant to proteolysis than CagA-M and CagA-R. Together with the fact that no accumulation of metastable fragments was observed, this suggested that fragment CagA-N folds into a single stable domain.

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