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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.

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State of XerA in solution at low protein concentration in 200 mM NaCl buffer.A. Dots: SAXS experimental data I(q) obtained on the SWING beamline. Red curve: calculated curve from the crystal structure with the missing residues added using BUNCH and SABBAC. B. Crystal structure superimposed on a typical envelope of the protein deduced from the SAXS experimental curve using the program GASBOR. The XerA monomer is in grey with the αMN helices in orange. C. Four models obtained using the program BUNCH from the crystal structure allowing the N-terminal domain to freely rotate. The grey structure is the crystal structure. All other models are in a variation of pink. For all models the agreement between the calculated curve and the experimental one is excellent (χ≈0.80).
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pone-0063010-g003: State of XerA in solution at low protein concentration in 200 mM NaCl buffer.A. Dots: SAXS experimental data I(q) obtained on the SWING beamline. Red curve: calculated curve from the crystal structure with the missing residues added using BUNCH and SABBAC. B. Crystal structure superimposed on a typical envelope of the protein deduced from the SAXS experimental curve using the program GASBOR. The XerA monomer is in grey with the αMN helices in orange. C. Four models obtained using the program BUNCH from the crystal structure allowing the N-terminal domain to freely rotate. The grey structure is the crystal structure. All other models are in a variation of pink. For all models the agreement between the calculated curve and the experimental one is excellent (χ≈0.80).

Mentions: In contrast to the closed structure of the apo-XerD monomer from Escherichia coli[15], XerA apo-monomers present a more open architecture in the crystal. The N- and C-terminal domains are well separated resulting in a marked C-shaped structure for the apo-XerA (Figure 1A). This also highlights the conformational freedom afforded by the extended linker. The relative orientation of the XerA N- and C-terminal domains is different from that of the XerD apo-structure (Figure 1A). This open conformation is also observed in solution at low concentration as revealed by SAXS experiments (Figure 3). Using the ab initio program GASBOR [44], which describes the scattering object as a chain of 292 dummy residues, an envelope of the protein can be deduced from the scattering curve. The typical envelope is extended and accommodates perfectly the crystal structure (Figure 3B). The theoretical scattering curve corresponding to the crystal structure was calculated and corrected for missing residues (8 at the N-terminal and 7 at the C-terminal), then fitted to the experimental curve (Figure 3A). The quality of the fit (χ = 0.80) indicates that the open conformation is present in solution and is not a consequence of crystal packing constraints. Nevertheless the SAXS curve is also compatible with models in which the XerA N-terminal domain possesses some rotational freedom (Figure 3C). Strikingly, the apo-XerA open structure is similar to the structures of Tyr-recombinases co-crystallized with DNA (Figure 2) and therefore seems to be in a configuration ready to bind DNA.


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)

State of XerA in solution at low protein concentration in 200 mM NaCl buffer.A. Dots: SAXS experimental data I(q) obtained on the SWING beamline. Red curve: calculated curve from the crystal structure with the missing residues added using BUNCH and SABBAC. B. Crystal structure superimposed on a typical envelope of the protein deduced from the SAXS experimental curve using the program GASBOR. The XerA monomer is in grey with the αMN helices in orange. C. Four models obtained using the program BUNCH from the crystal structure allowing the N-terminal domain to freely rotate. The grey structure is the crystal structure. All other models are in a variation of pink. For all models the agreement between the calculated curve and the experimental one is excellent (χ≈0.80).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063010-g003: State of XerA in solution at low protein concentration in 200 mM NaCl buffer.A. Dots: SAXS experimental data I(q) obtained on the SWING beamline. Red curve: calculated curve from the crystal structure with the missing residues added using BUNCH and SABBAC. B. Crystal structure superimposed on a typical envelope of the protein deduced from the SAXS experimental curve using the program GASBOR. The XerA monomer is in grey with the αMN helices in orange. C. Four models obtained using the program BUNCH from the crystal structure allowing the N-terminal domain to freely rotate. The grey structure is the crystal structure. All other models are in a variation of pink. For all models the agreement between the calculated curve and the experimental one is excellent (χ≈0.80).
Mentions: In contrast to the closed structure of the apo-XerD monomer from Escherichia coli[15], XerA apo-monomers present a more open architecture in the crystal. The N- and C-terminal domains are well separated resulting in a marked C-shaped structure for the apo-XerA (Figure 1A). This also highlights the conformational freedom afforded by the extended linker. The relative orientation of the XerA N- and C-terminal domains is different from that of the XerD apo-structure (Figure 1A). This open conformation is also observed in solution at low concentration as revealed by SAXS experiments (Figure 3). Using the ab initio program GASBOR [44], which describes the scattering object as a chain of 292 dummy residues, an envelope of the protein can be deduced from the scattering curve. The typical envelope is extended and accommodates perfectly the crystal structure (Figure 3B). The theoretical scattering curve corresponding to the crystal structure was calculated and corrected for missing residues (8 at the N-terminal and 7 at the C-terminal), then fitted to the experimental curve (Figure 3A). The quality of the fit (χ = 0.80) indicates that the open conformation is present in solution and is not a consequence of crystal packing constraints. Nevertheless the SAXS curve is also compatible with models in which the XerA N-terminal domain possesses some rotational freedom (Figure 3C). Strikingly, the apo-XerA open structure is similar to the structures of Tyr-recombinases co-crystallized with DNA (Figure 2) and therefore seems to be in a configuration ready to bind DNA.

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.

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Related in: MedlinePlus