Pseudomonas aeruginosa C-Terminal Processing Protease CtpA Assembles into a Hexameric Structure That Requires Activation by a Spiral-Shaped Lipoprotein-Binding Partner
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Evaluation Summary:
This paper demonstrates an inactive protease in Pseudomonas aeruginosa, CtpA, is regulated by am outer membrane lipoprotein LbcA. Using crystallization and EM strategies, they also provide a complex structure; however, the precise mechanism of regulation is speculative due to the flexible arrangement of protein domains.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
Carboxyl-terminal processing proteases (CTPs) are found in all three domains of life. In bacteria, some CTPs have been associated with virulence, raising the possibility that they could be therapeutic targets.
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Evaluation Summary:
This paper demonstrates an inactive protease in Pseudomonas aeruginosa, CtpA, is regulated by am outer membrane lipoprotein LbcA. Using crystallization and EM strategies, they also provide a complex structure; however, the precise mechanism of regulation is speculative due to the flexible arrangement of protein domains.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
Carboxy-terminal proteases, such as CtpA and CtpB, have been shown to be important for the normal functioning and the type 3 secretion system and virulence. The authors convincingly reveal the mechanism of how the inactive protease CtpA is activated by its substrate activator LbcA. Crystal structures of CtpA reveals a hexamer (trimer of dimers), with a Ser-Lys-Glu active site triad in an inactive conformation lacking proper H-bonding distance. Furthermore, in CtpA the substrate tunnel is blocked by a PDZ domain; these inactivating features are not observed in the active CtpB protein, revealing the structural rationale for protease inactivation with CtpA. Domain swapping facilitates oligomerization as does the C-terminal region extension not found in the active CtpB protease. The C-terminal region was shown …
Reviewer #1 (Public Review):
Carboxy-terminal proteases, such as CtpA and CtpB, have been shown to be important for the normal functioning and the type 3 secretion system and virulence. The authors convincingly reveal the mechanism of how the inactive protease CtpA is activated by its substrate activator LbcA. Crystal structures of CtpA reveals a hexamer (trimer of dimers), with a Ser-Lys-Glu active site triad in an inactive conformation lacking proper H-bonding distance. Furthermore, in CtpA the substrate tunnel is blocked by a PDZ domain; these inactivating features are not observed in the active CtpB protein, revealing the structural rationale for protease inactivation with CtpA. Domain swapping facilitates oligomerization as does the C-terminal region extension not found in the active CtpB protease. The C-terminal region was shown to influence the interface between the dimers of CtpA and the associate with the substrate activator LbcA. This was confirmed with mutagenesis studies. Crystal structure of LbcA revealed a spiral conformation consisting a 11 helical tetratricopeptide repeats, with an inner diameter of 3nm. The spiral is proposed by the authors to wrap around substrates. CryoEM of CtpA (An inactive serine mutant) with its substrate activator LbcA, revealed a stoichiometery of 6 CtpA to 3 LbcA. The LbcA indicated weak density but two conformations were observed where LbcA has either one or two contacts with CtpA, and importantly a rotation in the PDZ domain which initially blocked the active site. The N-terminus of LbcA was shown to be important for this association using pull down assays, and for substrate degradation both in vitro and in vivo. This, of course, is likely a complicated process, and the authors nicely present two different plausible models to support the activation of the CtpA by LbcA. This work will be of interest to readers in the protease field, and in particular those that function near membranes.
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Reviewer #2 (Public Review):
In this manuscript, Hsu et al. present structural insights into the substrate recruitment and proteolytic activation of the P. aeruginosa CTP protease CtpA. Notably, the authors conclude that CtpA remains in an inactivated state until interaction with adaptor protein LbcA, unlike CtpB, which can adopt the activated state without an adaptor protein. The authors show that CtpA alone assembles into a trimer of dimers, and the structures detail the interactions mediated by the NDR and CDR domain in hexamer formation. Biochemical mutagenesis was used to explore the functional importance of hexamerization and roles of these regions in oligomerization, which showed that disrupting hexamerization diminished, but did not completely abolish, CtpA's ability to degrade a model substrate PA1198. The crystal structure of …
Reviewer #2 (Public Review):
In this manuscript, Hsu et al. present structural insights into the substrate recruitment and proteolytic activation of the P. aeruginosa CTP protease CtpA. Notably, the authors conclude that CtpA remains in an inactivated state until interaction with adaptor protein LbcA, unlike CtpB, which can adopt the activated state without an adaptor protein. The authors show that CtpA alone assembles into a trimer of dimers, and the structures detail the interactions mediated by the NDR and CDR domain in hexamer formation. Biochemical mutagenesis was used to explore the functional importance of hexamerization and roles of these regions in oligomerization, which showed that disrupting hexamerization diminished, but did not completely abolish, CtpA's ability to degrade a model substrate PA1198. The crystal structure of LbcA confirmed that this adaptor protein contains 11 tetratricopeptide repeats, and that the authors were able to assemble a CtpA-LbcA complex through co-expression and pull-down. Using low resolution cryo-EM analyses and protease activity assays, the authors show that the N-terminal extension of LbcA is essential for its interaction with CtpA and activation of the protease. Based on these data, the authors conclude that protease activation is dependent on structural rearrangements induced by delivery of substrate to CtpA by the adaptor protein LbcA.
The main conclusions of the manuscript, which are that CtpA oligomerization and interaction with LbcA together mediate protease activity, is supported biochemically. While the authors propose an attractive model, in the absence of stronger structural data confirming the activated state in the presence of LbcA and substrate, the proposed mechanism remains very speculative.
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Reviewer #3 (Public Review):
In this study, Hsu et al. studied a protease system in Pseudomonas aeruginosa composed of CtpA (protease) and LbcA (am outer membrane lipoprotein) primarily using crystallization and EM strategies. They find that the protease CtpA alone forms an inactive trimer of dimers (hexamer) that limits substrate access to prevent nonspecific protein degradation, and that the N- and C- termini of CtpA function in both dimerization and CtpA protease activity. The CtpA alone is inactive unless its partner LbcA is present. They then solved the crystal structure of LbcA. In the presence of both CtpA and LbcA, they observed by cryo-EM that three LbcA molecules, each with a spiral structure, bind a CtpA hexamer and potentially induce conformational changes in CtpA, switching on CtpA. Moreover, additional data indicated that …
Reviewer #3 (Public Review):
In this study, Hsu et al. studied a protease system in Pseudomonas aeruginosa composed of CtpA (protease) and LbcA (am outer membrane lipoprotein) primarily using crystallization and EM strategies. They find that the protease CtpA alone forms an inactive trimer of dimers (hexamer) that limits substrate access to prevent nonspecific protein degradation, and that the N- and C- termini of CtpA function in both dimerization and CtpA protease activity. The CtpA alone is inactive unless its partner LbcA is present. They then solved the crystal structure of LbcA. In the presence of both CtpA and LbcA, they observed by cryo-EM that three LbcA molecules, each with a spiral structure, bind a CtpA hexamer and potentially induce conformational changes in CtpA, switching on CtpA. Moreover, additional data indicated that the N-terminal helices of LbcA, especially H1, are involved in CtpA binding and protease system activation. Overall, this paper describes the potential mechanism of CtpA-LbcA complex and reports the most essential regions in both proteins that play indispensable roles. In general, conclusions and predictions in this paper were supported by the data obtained from properly designed biochemical and biophysical experiments. However, a few points are still not clear enough and could be improved.
1. In the section that describes the 3:6 active protease complex, it would be better to explain why the protease dead mutant CtpA (S302A) is used.
2. Even though the overall data analysis and predications of CtpA-LbcA complex make sense, an 8 Å resolution of the complex and the high flexibility of the PDZ domain of CtpA still bring uncertainty. Moreover, for the movements reported in the PDZ region of CtpA upon LbcA binding, please make it clear if it is observed in both conformer I and II, or only in conformer II, the active mode. The current descriptions in results, discussions and figures are ambiguous. Also, other than the twist in PDZ, there is no other changes in CtpA from model fitting? As shown in Fig 5 c, between the two conformers, the difference of the LbcA N-terminal densities at the binding interface is obvious. Changes in CtpA NDR region, which is involved in the binding interface; or in the CtpA cap region, which is associated with the catalytic features and is pushed downward by LbcA in conformer II, should be interesting.
3. The consistency of the texts and figure legends on which LbcA construct is used in the protease complex should be double checked. Same for the descriptions of the LbcA structure. A four-helix bundle is delineated, but the helices in text (H1H2, H3H4) and figures (H1H4, H2H3) are different. In addition, the four-helix bundle is shown in Figure 4 d, but the design of Figure 4 d does not explain it well. Actually, supplementary figure 3 C and D make this point more understandable. Also, for Figure 4 c, it would be better to mark all the helices, which could directly show TPR-A and B line the outer and inner surfaces and explain the hydrophobic and hydrophilic idea. Figure 5 d could be improved since the current version is not adequate to elucidate how PDZ gets shifted and rotated in a big complex. Besides, the color scheme in figures may also cause some confusion, such as in Figure 1 and 2, the same colors are used to represent different concepts. Since this paper is rich in content, it is necessary to keep consistency and avoid potentially misleading readers.
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