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Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance.

Lillington JE, Lovett JE, Johnson S, Roversi P, Timmel CR, Lea SM - J. Mol. Biol. (2010)

Bottom Line: One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG.This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets.The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.

View Article: PubMed Central - PubMed

Affiliation: Inorganic Chemistry Laboratory, University of Oxford, OX1 3QR, UK.

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Impact of IpgB1 binding on Spa15 DEER. (a) DEER trace for Spa15:IpgB1. Note: Modulation depths between Fig. 1a and panel (a) of this figure are not directly comparable due to different technical conditions when the samples were measured. (b) DEER-derived distances for Spa15 dimer (black) compared to Spa15:IpgB1 (blue) show no significant difference in distance between the Cys19 residues of Spa15 when the effector is bound or unbound. (c) Part of the InvB:SipA structure (PDB 2FM8) with spin-labeled Spa15 (grey surface) replacing InvB. SipA (red ribbon) occludes the binding pocket from the spin label. MTSL is shown in green (atom coloured stick), clashing with SipA.
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f0015: Impact of IpgB1 binding on Spa15 DEER. (a) DEER trace for Spa15:IpgB1. Note: Modulation depths between Fig. 1a and panel (a) of this figure are not directly comparable due to different technical conditions when the samples were measured. (b) DEER-derived distances for Spa15 dimer (black) compared to Spa15:IpgB1 (blue) show no significant difference in distance between the Cys19 residues of Spa15 when the effector is bound or unbound. (c) Part of the InvB:SipA structure (PDB 2FM8) with spin-labeled Spa15 (grey surface) replacing InvB. SipA (red ribbon) occludes the binding pocket from the spin label. MTSL is shown in green (atom coloured stick), clashing with SipA.

Mentions: We were interested in whether DEER could detect a structural change upon binding of the effector IpgB1 to Spa15. The co-purification of Spa15:IpgB1complex at 2:1 ratio was confirmed using multi-angle laser light scattering (data not shown), showing that IpgB1 has no effect on the stoichiometry of the dimeric Spa15. The complex was spin labeled; mass spectrometry confirmed the spin labeling of Spa15 Cys19 in this complex, but not IpgB1 (Cys49 and Cys75) (data not shown). This inability of IpgB1's two cysteine residues to be spin labeled demonstrates their surface inaccessibility in this complex. Spin labeling of the Spa15:IpgB1 complex had no significant effect (± 0.1 nm) on the major DEER distance relative to that of the Spa15 dimer (Fig. 3a and b). Satellite peaks were again observable due to the high concentration conditions.


Shigella flexneri Spa15 crystal structure verified in solution by double electron electron resonance.

Lillington JE, Lovett JE, Johnson S, Roversi P, Timmel CR, Lea SM - J. Mol. Biol. (2010)

Impact of IpgB1 binding on Spa15 DEER. (a) DEER trace for Spa15:IpgB1. Note: Modulation depths between Fig. 1a and panel (a) of this figure are not directly comparable due to different technical conditions when the samples were measured. (b) DEER-derived distances for Spa15 dimer (black) compared to Spa15:IpgB1 (blue) show no significant difference in distance between the Cys19 residues of Spa15 when the effector is bound or unbound. (c) Part of the InvB:SipA structure (PDB 2FM8) with spin-labeled Spa15 (grey surface) replacing InvB. SipA (red ribbon) occludes the binding pocket from the spin label. MTSL is shown in green (atom coloured stick), clashing with SipA.
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Related In: Results  -  Collection

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Show All Figures
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f0015: Impact of IpgB1 binding on Spa15 DEER. (a) DEER trace for Spa15:IpgB1. Note: Modulation depths between Fig. 1a and panel (a) of this figure are not directly comparable due to different technical conditions when the samples were measured. (b) DEER-derived distances for Spa15 dimer (black) compared to Spa15:IpgB1 (blue) show no significant difference in distance between the Cys19 residues of Spa15 when the effector is bound or unbound. (c) Part of the InvB:SipA structure (PDB 2FM8) with spin-labeled Spa15 (grey surface) replacing InvB. SipA (red ribbon) occludes the binding pocket from the spin label. MTSL is shown in green (atom coloured stick), clashing with SipA.
Mentions: We were interested in whether DEER could detect a structural change upon binding of the effector IpgB1 to Spa15. The co-purification of Spa15:IpgB1complex at 2:1 ratio was confirmed using multi-angle laser light scattering (data not shown), showing that IpgB1 has no effect on the stoichiometry of the dimeric Spa15. The complex was spin labeled; mass spectrometry confirmed the spin labeling of Spa15 Cys19 in this complex, but not IpgB1 (Cys49 and Cys75) (data not shown). This inability of IpgB1's two cysteine residues to be spin labeled demonstrates their surface inaccessibility in this complex. Spin labeling of the Spa15:IpgB1 complex had no significant effect (± 0.1 nm) on the major DEER distance relative to that of the Spa15 dimer (Fig. 3a and b). Satellite peaks were again observable due to the high concentration conditions.

Bottom Line: One of these effectors is IpgB1, a mimic of the human Ras-like Rho guanosine triphosphatase RhoG.This distance is explained by determining the crystal structure of the spin-labeled Spa15 where labels are seen to be buried in hydrophobic pockets.The double electron electron resonance experiment on the Spa15 complex with IpgB1 shows that IpgB1 does not bind Spa15 in the same way as is seen in the homologous Salmonella sp. chaperone:effector complex InvB:SipA.

View Article: PubMed Central - PubMed

Affiliation: Inorganic Chemistry Laboratory, University of Oxford, OX1 3QR, UK.

Show MeSH
Related in: MedlinePlus