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Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Srikannathasan V, English G, Bui NK, Trunk K, O'Rourke PE, Rao VA, Vollmer W, Coulthurst SJ, Hunter WN - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized.Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness.Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.

ABSTRACT
Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

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Primary, secondary and tertiary structure of Rap2a. (a) The amino-acid sequence with assigned secondary structure and helices numbered. Residues involved in disulfide-bond formation are coloured yellow and residues involved in subunit–subunit interactions are shown on a blue background. (b) Cartoon representation of the Rap1a dimer with subunits coloured brown and green. The disulfides and the N- and C-termini are labelled. (c) Cartoon representation of Rap2b (pink) and EcTai4 (grey) superimposed on Rap2a (green). Asp54 and Asp47 are involved in the hydrogen-bond network in the interface in EcTai4 and are absolutely conserved in Rap2b; in Rap2a, Asp47 is replaced by Ser48. Arg40 and Glu74 (replaced by Val41 and Thr75, respectively, in Rap2a) have been shown to play a major role in binding to EcTae4; both of these residues are also conserved in Rap2b but not in Rap2a.
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fig6: Primary, secondary and tertiary structure of Rap2a. (a) The amino-acid sequence with assigned secondary structure and helices numbered. Residues involved in disulfide-bond formation are coloured yellow and residues involved in subunit–subunit interactions are shown on a blue background. (b) Cartoon representation of the Rap1a dimer with subunits coloured brown and green. The disulfides and the N- and C-termini are labelled. (c) Cartoon representation of Rap2b (pink) and EcTai4 (grey) superimposed on Rap2a (green). Asp54 and Asp47 are involved in the hydrogen-bond network in the interface in EcTai4 and are absolutely conserved in Rap2b; in Rap2a, Asp47 is replaced by Ser48. Arg40 and Glu74 (replaced by Val41 and Thr75, respectively, in Rap2a) have been shown to play a major role in binding to EcTae4; both of these residues are also conserved in Rap2b but not in Rap2a.

Mentions: The Rap2a subunit displays a compact globular structure of five α-helices with an extended loop linking α3 to α4 (Figs. 6 ▶a and 6 ▶b). A disulfide bond between Cys42 and Cys102 links α1 to α4 and interactions of other side-chain groups on these elements of secondary structure help to create the helical bundle fold and in particular to align α2. Together, α1, α2, the α2–α3 loop and α4 form the dimer interface, giving rise to a twofold NCS axis, and a combination of hydrogen-bonding, salt-bridge and van der Waals interactions serve to stabilize the association. Main-chain hydrogen-bonding contributions come from the amides of Ser48, Ala49, Met97, Thr98 and Met99 on both chains. Side-chain contributions come from the hydroxyl groups of Tyr28 and Tyr47 and the carboxylates of Glu51, Asp55 and Asp104. Several solvent-mediated contacts also serve to link functional groups on partner subunits (not shown). The side chains of Leu39, Ile43, Tyr47, Val52, Met99 and Ile103 are involved in van der Waals interactions with the partner subunit to stabilize the dimer.


Structural basis for type VI secreted peptidoglycan DL-endopeptidase function, specificity and neutralization in Serratia marcescens.

Srikannathasan V, English G, Bui NK, Trunk K, O'Rourke PE, Rao VA, Vollmer W, Coulthurst SJ, Hunter WN - Acta Crystallogr. D Biol. Crystallogr. (2013)

Primary, secondary and tertiary structure of Rap2a. (a) The amino-acid sequence with assigned secondary structure and helices numbered. Residues involved in disulfide-bond formation are coloured yellow and residues involved in subunit–subunit interactions are shown on a blue background. (b) Cartoon representation of the Rap1a dimer with subunits coloured brown and green. The disulfides and the N- and C-termini are labelled. (c) Cartoon representation of Rap2b (pink) and EcTai4 (grey) superimposed on Rap2a (green). Asp54 and Asp47 are involved in the hydrogen-bond network in the interface in EcTai4 and are absolutely conserved in Rap2b; in Rap2a, Asp47 is replaced by Ser48. Arg40 and Glu74 (replaced by Val41 and Thr75, respectively, in Rap2a) have been shown to play a major role in binding to EcTae4; both of these residues are also conserved in Rap2b but not in Rap2a.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852654&req=5

fig6: Primary, secondary and tertiary structure of Rap2a. (a) The amino-acid sequence with assigned secondary structure and helices numbered. Residues involved in disulfide-bond formation are coloured yellow and residues involved in subunit–subunit interactions are shown on a blue background. (b) Cartoon representation of the Rap1a dimer with subunits coloured brown and green. The disulfides and the N- and C-termini are labelled. (c) Cartoon representation of Rap2b (pink) and EcTai4 (grey) superimposed on Rap2a (green). Asp54 and Asp47 are involved in the hydrogen-bond network in the interface in EcTai4 and are absolutely conserved in Rap2b; in Rap2a, Asp47 is replaced by Ser48. Arg40 and Glu74 (replaced by Val41 and Thr75, respectively, in Rap2a) have been shown to play a major role in binding to EcTae4; both of these residues are also conserved in Rap2b but not in Rap2a.
Mentions: The Rap2a subunit displays a compact globular structure of five α-helices with an extended loop linking α3 to α4 (Figs. 6 ▶a and 6 ▶b). A disulfide bond between Cys42 and Cys102 links α1 to α4 and interactions of other side-chain groups on these elements of secondary structure help to create the helical bundle fold and in particular to align α2. Together, α1, α2, the α2–α3 loop and α4 form the dimer interface, giving rise to a twofold NCS axis, and a combination of hydrogen-bonding, salt-bridge and van der Waals interactions serve to stabilize the association. Main-chain hydrogen-bonding contributions come from the amides of Ser48, Ala49, Met97, Thr98 and Met99 on both chains. Side-chain contributions come from the hydroxyl groups of Tyr28 and Tyr47 and the carboxylates of Glu51, Asp55 and Asp104. Several solvent-mediated contacts also serve to link functional groups on partner subunits (not shown). The side chains of Leu39, Ile43, Tyr47, Val52, Met99 and Ile103 are involved in van der Waals interactions with the partner subunit to stabilize the dimer.

Bottom Line: Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized.Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness.Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.

ABSTRACT
Some Gram-negative bacteria target their competitors by exploiting the type VI secretion system to extrude toxic effector proteins. To prevent self-harm, these bacteria also produce highly specific immunity proteins that neutralize these antagonistic effectors. Here, the peptidoglycan endopeptidase specificity of two type VI secretion-system-associated effectors from Serratia marcescens is characterized. These small secreted proteins, Ssp1 and Ssp2, cleave between γ-D-glutamic acid and L-meso-diaminopimelic acid with different specificities. Ssp2 degrades the acceptor part of cross-linked tetratetrapeptides. Ssp1 displays greater promiscuity and cleaves monomeric tripeptides, tetrapeptides and pentapeptides and dimeric tetratetra and tetrapenta muropeptides on both the acceptor and donor strands. Functional assays confirm the identity of a catalytic cysteine in these endopeptidases and crystal structures provide information on the structure-activity relationships of Ssp1 and, by comparison, of related effectors. Functional assays also reveal that neutralization of these effectors by their cognate immunity proteins, which are called resistance-associated proteins (Raps), contributes an essential role to cell fitness. The structures of two immunity proteins, Rap1a and Rap2a, responsible for the neutralization of Ssp1 and Ssp2-like endopeptidases, respectively, revealed two distinct folds, with that of Rap1a not having previously been observed. The structure of the Ssp1-Rap1a complex revealed a tightly bound heteromeric assembly with two effector molecules flanking a Rap1a dimer. A highly effective steric block of the Ssp1 active site forms the basis of effector neutralization. Comparisons with Ssp2-Rap2a orthologues suggest that the specificity of these immunity proteins for neutralizing effectors is fold-dependent and that in cases where the fold is conserved sequence differences contribute to the specificity of effector-immunity protein interactions.

Show MeSH
Related in: MedlinePlus