Limits...
The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

Serre MC, El Arnaout T, Brooks MA, Durand D, Lisboa J, Lazar N, Raynal B, van Tilbeurgh H, Quevillon-Cheruel S - PLoS ONE (2013)

Bottom Line: Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates.Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis.We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France. marie-claude.serre@igmors.u-psud.fr

ABSTRACT
Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

Show MeSH

Related in: MedlinePlus

Structure of XerA.A. Overall structure of the XerA and XerD apo-monomers. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan. The β2–β3 loop is in pink and the C-terminal αMN helices are orange. The C-terminal His-tag is not visible in the electronic density reflecting its mobility. C. 2Fo-Fc electron density map (blue). The backbone of the protein is represented by ribbons (green and red). The distance between the two last visible amino-acids is indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646895&req=5

pone-0063010-g001: Structure of XerA.A. Overall structure of the XerA and XerD apo-monomers. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan. The β2–β3 loop is in pink and the C-terminal αMN helices are orange. The C-terminal His-tag is not visible in the electronic density reflecting its mobility. C. 2Fo-Fc electron density map (blue). The backbone of the protein is represented by ribbons (green and red). The distance between the two last visible amino-acids is indicated.

Mentions: Although diffraction-quality crystals of native XerA were obtained, they could not be reproduced when using the Seleno-Methionine labeled protein. Therefore the structure of full-length XerA was solved by molecular replacement in two stages, using the separate domains independently as search models. Firstly, we used the N-terminal domain of XerA that we solved separately at 3.0 Å resolution (data not shown) as the search model. Secondly, we used the XerD catalytic domain as the search model to solve the XerA C-terminal domain. The final model has a resolution of 3.0 Å and displays good stereochemistry (Table 1). The electron density between H252 and T258 is weak but sufficiently continuous to perceive the link (represented by a broken line in Figure 1). A composite omit map contoured at 0.5 sigma is presented in Figure 1B, and illustrates that the chain does appear to be continuous in this region, albeit with very weak electron density. A search for structurally related proteins in the PDB (Table S1) using the DALI algorithm [41] revealed that the most closely related structure to the N-terminal domain of XerA (residues 9 to 90) is the core-binding domain of λ-Int [42]. Superimposition of these domains gives an r.m.s.d. of 2.22 Å of Cα atoms, with 90% of residues aligned and 12% sequence identity. The most similar structure to the XerA C-terminal domain (residues 107 to 279) is the catalytic domain of IntIA [12], with an r.m.s.d. of 2.3 Å over 152 aligned residues and 41% sequence identity.


The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

Serre MC, El Arnaout T, Brooks MA, Durand D, Lisboa J, Lazar N, Raynal B, van Tilbeurgh H, Quevillon-Cheruel S - PLoS ONE (2013)

Structure of XerA.A. Overall structure of the XerA and XerD apo-monomers. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan. The β2–β3 loop is in pink and the C-terminal αMN helices are orange. The C-terminal His-tag is not visible in the electronic density reflecting its mobility. C. 2Fo-Fc electron density map (blue). The backbone of the protein is represented by ribbons (green and red). The distance between the two last visible amino-acids is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646895&req=5

pone-0063010-g001: Structure of XerA.A. Overall structure of the XerA and XerD apo-monomers. Colour code of active site residues: Arg: magenta, Lys: dark blue, His: yellow, Tyr: red. The sulfate ion present in the active site is in cyan. The β2–β3 loop is in pink and the C-terminal αMN helices are orange. The C-terminal His-tag is not visible in the electronic density reflecting its mobility. C. 2Fo-Fc electron density map (blue). The backbone of the protein is represented by ribbons (green and red). The distance between the two last visible amino-acids is indicated.
Mentions: Although diffraction-quality crystals of native XerA were obtained, they could not be reproduced when using the Seleno-Methionine labeled protein. Therefore the structure of full-length XerA was solved by molecular replacement in two stages, using the separate domains independently as search models. Firstly, we used the N-terminal domain of XerA that we solved separately at 3.0 Å resolution (data not shown) as the search model. Secondly, we used the XerD catalytic domain as the search model to solve the XerA C-terminal domain. The final model has a resolution of 3.0 Å and displays good stereochemistry (Table 1). The electron density between H252 and T258 is weak but sufficiently continuous to perceive the link (represented by a broken line in Figure 1). A composite omit map contoured at 0.5 sigma is presented in Figure 1B, and illustrates that the chain does appear to be continuous in this region, albeit with very weak electron density. A search for structurally related proteins in the PDB (Table S1) using the DALI algorithm [41] revealed that the most closely related structure to the N-terminal domain of XerA (residues 9 to 90) is the core-binding domain of λ-Int [42]. Superimposition of these domains gives an r.m.s.d. of 2.22 Å of Cα atoms, with 90% of residues aligned and 12% sequence identity. The most similar structure to the XerA C-terminal domain (residues 107 to 279) is the catalytic domain of IntIA [12], with an r.m.s.d. of 2.3 Å over 152 aligned residues and 41% sequence identity.

Bottom Line: Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates.Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis.We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France. marie-claude.serre@igmors.u-psud.fr

ABSTRACT
Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i) repositioning of the catalytic Tyr in the active site in cis and (ii) dimer stabilisation via αN contacts in trans between monomers.

Show MeSH
Related in: MedlinePlus