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Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

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A and B, SPR sensorgram and resulting affinity fit for HrcN (type III export ATPase) binding to biotinylated cdG. C and D, SPR sensorgram and resulting affinity fit for ClpB2 (Type VI export ATPase) binding to biotinylated cdG. In both cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, and 10 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad (Table 3).
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Figure 4: A and B, SPR sensorgram and resulting affinity fit for HrcN (type III export ATPase) binding to biotinylated cdG. C and D, SPR sensorgram and resulting affinity fit for ClpB2 (Type VI export ATPase) binding to biotinylated cdG. In both cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, and 10 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad (Table 3).

Mentions: The export apparatus of the bacterial flagellum is closely related to that of the T3SS, with both complexes sharing a common ancestor (8). Furthermore, cdG has been associated with the control of T3SS function in the opportunistic pathogen Pseudomonas aeruginosa (64, 65), although the mechanism of this regulation is currently unclear. In light of this, our data for FliI-cdG binding implicate the T3SS export ATPase HrcN as a further potential cdG-binding target. To test this, we purified the full-length, His-tagged protein from Pto DC3000 (HrcNHis) and examined cdG binding using SPR. As predicted, HrcNHis also bound strongly to cdG, with a dissociation constant of 3.2 ± 0.2 μm (Fig. 4, A and B). The type VI secretion system export ATPase (ClpB2) is far more distantly related to FliI, in terms of both primary sequence and organization of the ATPase subunits within the type VI secretion complex (66). Nonetheless, as ClpB2 is a rotary ATPase and type VI secretion is known to be under reciprocal, cdG-linked control with type III secretion (65), full-length ClpB2 (ClpB2His) was purified and tested for cdG binding. To our surprise, ClpB2 also displayed strong, concentration-dependent binding to the cdG with a physiologically relevant binding affinity of 9.5 ± 0.5 μm (Fig. 4, C and D). These data strongly suggest that binding to the cdG second messenger is a widespread characteristic across diverse rotary ATPase export proteins.


Bacterial rotary export ATPases are allosterically regulated by the nucleotide second messenger cyclic-di-GMP.

Trampari E, Stevenson CE, Little RH, Wilhelm T, Lawson DM, Malone JG - J. Biol. Chem. (2015)

A and B, SPR sensorgram and resulting affinity fit for HrcN (type III export ATPase) binding to biotinylated cdG. C and D, SPR sensorgram and resulting affinity fit for ClpB2 (Type VI export ATPase) binding to biotinylated cdG. In both cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, and 10 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad (Table 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591828&req=5

Figure 4: A and B, SPR sensorgram and resulting affinity fit for HrcN (type III export ATPase) binding to biotinylated cdG. C and D, SPR sensorgram and resulting affinity fit for ClpB2 (Type VI export ATPase) binding to biotinylated cdG. In both cases, a range of protein concentrations was used (0.625, 1.25, 2.5, 5, and 10 μm), and concentration replicates were included as appropriate together with buffer only and BSA controls. The protein binding and dissociation phases for all sensorgrams are shown. For the affinity fits, binding responses were measured 4 s before the end of the injection, and KD values for each protein were calculated using the BiaEvaluation software and confirmed by GraphPad (Table 3).
Mentions: The export apparatus of the bacterial flagellum is closely related to that of the T3SS, with both complexes sharing a common ancestor (8). Furthermore, cdG has been associated with the control of T3SS function in the opportunistic pathogen Pseudomonas aeruginosa (64, 65), although the mechanism of this regulation is currently unclear. In light of this, our data for FliI-cdG binding implicate the T3SS export ATPase HrcN as a further potential cdG-binding target. To test this, we purified the full-length, His-tagged protein from Pto DC3000 (HrcNHis) and examined cdG binding using SPR. As predicted, HrcNHis also bound strongly to cdG, with a dissociation constant of 3.2 ± 0.2 μm (Fig. 4, A and B). The type VI secretion system export ATPase (ClpB2) is far more distantly related to FliI, in terms of both primary sequence and organization of the ATPase subunits within the type VI secretion complex (66). Nonetheless, as ClpB2 is a rotary ATPase and type VI secretion is known to be under reciprocal, cdG-linked control with type III secretion (65), full-length ClpB2 (ClpB2His) was purified and tested for cdG binding. To our surprise, ClpB2 also displayed strong, concentration-dependent binding to the cdG with a physiologically relevant binding affinity of 9.5 ± 0.5 μm (Fig. 4, C and D). These data strongly suggest that binding to the cdG second messenger is a widespread characteristic across diverse rotary ATPase export proteins.

Bottom Line: The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro.Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits.Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins.

View Article: PubMed Central - PubMed

Affiliation: From the Molecular Microbiology Department and.

Show MeSH
Related in: MedlinePlus