PcrG protects the two long helical oligomerization domains of PcrV, by an interaction mediated by the intramolecular coiled-coil region of PcrG

BMC Structural Biology

Saumen Datta¹  Supratim Dey¹  Urmisha Das¹  Abhishek Basu¹ 

[1] Structural Biology and Bioinformatics division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata 700032 West Bengal, India

关键词: Molecular docking;    Surface plasmon resonance and protein-protein interaction;    Deletion mutants;    Intramolecular coiled-coil;    Reversal of oligomerization;    MS/MS sequence analysis;    Protease protected fragment;    Homology model;    Dynamic light scattering and elongated conformation;    Functional translocon;    Regulation of TTSS;   

Others  :  793253
DOI  :  10.1186/1472-6807-14-5

 received in 2013-09-25, accepted in 2014-01-17,  发布年份 2014

【 摘 要 】

Background

PcrV is a hydrophilic translocator of type three secretion system (TTSS) and a structural component of the functional translocon. C-terminal helix of PcrV is essential for its oligomerization at the needle tip. Conformational changes within PcrV regulate the effector translocation. PcrG is a cytoplasmic regulator of TTSS and forms a high affinity complex with PcrV. C-terminal residues of PcrG control the effector secretion.

Result

Both PcrV and PcrG-PcrV complex exhibit elongated conformation like their close homologs LcrV and LcrG-LcrV complex. The homology model of PcrV depicts a dumbbell shaped structure with N and C-terminal globular domains. The grip of the dumbbell is formed by two long helices (helix-7 and 12), which show high level of conservation both structurally and evolutionary. PcrG specifically protects a region of PcrV extending from helix-12 to helix-7, and encompassing the C-terminal globular domain. This fragment ∆PcrV_(128–294) interacts with PcrG with high affinity, comparable to the wild type interaction. Deletion of N-terminal globular domain leads to the oligomerization of PcrV, but PcrG restores the monomeric state of PcrV by forming a heterodimeric complex. The N-terminal globular domain (∆PcrV_(1–127)) does not interact with PcrG but maintains its monomeric state. Interaction affinities of various domains of PcrV with PcrG illustrates that helix-12 is the key mediator of PcrG-PcrV interaction, supported by helix-7. Bioinformatic analysis and study with our deletion mutant ∆PcrG_(13–72) revealed that the first predicted intramolecular coiled-coil domain of PcrG contains the PcrV interaction site. However, 12 N-terminal amino acids of PcrG play an indirect role in PcrG-PcrV interaction, as their deletion causes 40-fold reduction in binding affinity and changes the kinetic parameters of interaction. ∆PcrG_(13–72) fits within the groove formed between the two globular domains of PcrV, through hydrophobic interaction.

Conclusion

PcrG interacts with PcrV through its intramolecular coiled-coil region and masks the domains responsible for oligomerization of PcrV at the needle tip. Also, PcrG could restore the monomeric state of oligomeric PcrV. Therefore, PcrG prevents the premature oligomerization of PcrV and maintains its functional state within the bacterial cytoplasm, which is a pre-requisite for formation of the functional translocon.

【 授权许可】

   
2014 Basu et al.; licensee BioMed Central Ltd.

【 图 表 】

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