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The metallo-beta-lactamase/beta-CASP domain of Artemis constitutes the catalytic core for V(D)J recombination.

Poinsignon C, Moshous D, Callebaut I, de Chasseval R, Villey I, de Villartay JP - J. Exp. Med. (2004)

Bottom Line: Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates.Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity.The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

View Article: PubMed Central - PubMed

Affiliation: Développement Normal et Pathologique de Système Immunitaire, INSERM U429, Hôpital Necker Enfants Malades, 75015 Paris, France.

ABSTRACT
The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-beta-lactamase (beta-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the beta-Lact homology domain, to which is appended (b) the beta-CASP region, specific of members of the beta-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

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V(D)J recombinase activity of in vitro–generated Artemis mutants. (A) All mutants are expressed in 293T cells and retain their capacity to interact with DNA-PKcs. (B) FACS® analysis of GUETEL/RSS cells transiently transfected with RAG1, RAG2, and the various Artemis mutants. The percent of recombination refers to the frequency of EGFP+ cells among the CD4+ cells. (C) Integrated results of four experiments showing the relative V(D)J recombination activity of the mutants relative to the recombination frequency using GST-Arte. (D) Hypothetical model for the structure of the catalytic site of Artemis. This model is based on the structure of the B. cereus and S. maltophilia β-Lacts as adapted from Wang et al. (reference 14; PDB codes: 1BC2 and 1SML, respectively). Gray and red spheres indicate the position of zinc and water oxygen atoms, respectively. Shaded areas correspond to the two Zn(II)-binding domains. The green star indicates the position of the fifth Zn2 ligand, which may correspond to a water molecule, or to a conserved amino acid of the β-CASP region (possibly D165).
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fig4: V(D)J recombinase activity of in vitro–generated Artemis mutants. (A) All mutants are expressed in 293T cells and retain their capacity to interact with DNA-PKcs. (B) FACS® analysis of GUETEL/RSS cells transiently transfected with RAG1, RAG2, and the various Artemis mutants. The percent of recombination refers to the frequency of EGFP+ cells among the CD4+ cells. (C) Integrated results of four experiments showing the relative V(D)J recombination activity of the mutants relative to the recombination frequency using GST-Arte. (D) Hypothetical model for the structure of the catalytic site of Artemis. This model is based on the structure of the B. cereus and S. maltophilia β-Lacts as adapted from Wang et al. (reference 14; PDB codes: 1BC2 and 1SML, respectively). Gray and red spheres indicate the position of zinc and water oxygen atoms, respectively. Shaded areas correspond to the two Zn(II)-binding domains. The green star indicates the position of the fifth Zn2 ligand, which may correspond to a water molecule, or to a conserved amino acid of the β-CASP region (possibly D165).

Mentions: The catalytic residues that define the two Zn(II)-binding pockets of class B β-Lacts concord to a highly conserved consensus signature: [HxHxD]…[H]…[C]…[H] (14). As most of these residues are conserved in Artemis (Fig. 1 B), we analyzed their role in Artemis function through in vitro mutagenesis. All engineered mutants were normally expressed and retained the capacity to interact with DNA-PKcs as judged by coimmunoprecipitation assay (Fig. 4 A). One representative experiment (Fig. 4 B) demonstrates that the basal V(D)J recombination activity in GUETEL/RSS cells (0.12%) is fully complemented by both Artemis (0.99%) or GST-Artemis (1.25%) expression constructs despite the previously reported reduced activity of a GST-Artemis fusion protein in vitro (10). GST-Artemis alone did not induce V(D)J recombination (0.01%) as expected. The integrated results of four experiments is shown in Fig. 4 C. Based on sequence analysis, the H33/H35/H115 triad in Artemis was predicted to correspond to the ligands of the first zinc (Zn1) of the binuclear Zn(II) center (Fig. 4 D). Accordingly, replacement of these residues with alanine strongly compromises the Artemis activity in all three mutants (Fig. 4 C). As a control, the mutant H151A, which affects a nonconserved His residue outside of the putative catalytic site, does not alter Artemis function. In the structure of class B enzymes as shown for Bacillus cereus in Fig. 4 D, the second zinc (Zn2) is coordinated by an aspartic acid, a histidine and a cysteine, and by two water molecules. The Asp residue is conserved in Artemis and its replacement by alanine (D37A) abolishes Artemis' activity. The Cys (Cys168 in B. cereus) is conserved in class B β-Lacts except for L1 enzyme of Stenotrophomonas maltophilia where it is replaced by a Ser at position 185, which is not a ligand of the zinc ion (14, 19). Instead, His89 provides the fifth Zn2 ligation in the L1 structure, next to the Asp88 residue in motif II (Figs. 1 B and 4 D). The Cys residue is not present in Artemis either, where Asp136 replaces it. Although this residue could substitute for the Cys in Zinc binding, it is interesting to note that Artemis also contains a His at position 38 in motif II that could participate in the Zn(II) coordination (Figs. 1 B and 4 D). To discriminate between these two possibilities, we analyzed the function of Artemis carrying alanine mutations at these two positions. Although H38A mutant is fully active, D136A has lost most of the catalytic activity (Fig. 4 C). Therefore, this result suggests that the putative second Zn(II) center of the Artemis catalytic site would adopt a structure similar to that of the B. cereus enzyme with Asp136 substituting the conserved Cys (Figs. 1 B and 4 D).


The metallo-beta-lactamase/beta-CASP domain of Artemis constitutes the catalytic core for V(D)J recombination.

Poinsignon C, Moshous D, Callebaut I, de Chasseval R, Villey I, de Villartay JP - J. Exp. Med. (2004)

V(D)J recombinase activity of in vitro–generated Artemis mutants. (A) All mutants are expressed in 293T cells and retain their capacity to interact with DNA-PKcs. (B) FACS® analysis of GUETEL/RSS cells transiently transfected with RAG1, RAG2, and the various Artemis mutants. The percent of recombination refers to the frequency of EGFP+ cells among the CD4+ cells. (C) Integrated results of four experiments showing the relative V(D)J recombination activity of the mutants relative to the recombination frequency using GST-Arte. (D) Hypothetical model for the structure of the catalytic site of Artemis. This model is based on the structure of the B. cereus and S. maltophilia β-Lacts as adapted from Wang et al. (reference 14; PDB codes: 1BC2 and 1SML, respectively). Gray and red spheres indicate the position of zinc and water oxygen atoms, respectively. Shaded areas correspond to the two Zn(II)-binding domains. The green star indicates the position of the fifth Zn2 ligand, which may correspond to a water molecule, or to a conserved amino acid of the β-CASP region (possibly D165).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211804&req=5

fig4: V(D)J recombinase activity of in vitro–generated Artemis mutants. (A) All mutants are expressed in 293T cells and retain their capacity to interact with DNA-PKcs. (B) FACS® analysis of GUETEL/RSS cells transiently transfected with RAG1, RAG2, and the various Artemis mutants. The percent of recombination refers to the frequency of EGFP+ cells among the CD4+ cells. (C) Integrated results of four experiments showing the relative V(D)J recombination activity of the mutants relative to the recombination frequency using GST-Arte. (D) Hypothetical model for the structure of the catalytic site of Artemis. This model is based on the structure of the B. cereus and S. maltophilia β-Lacts as adapted from Wang et al. (reference 14; PDB codes: 1BC2 and 1SML, respectively). Gray and red spheres indicate the position of zinc and water oxygen atoms, respectively. Shaded areas correspond to the two Zn(II)-binding domains. The green star indicates the position of the fifth Zn2 ligand, which may correspond to a water molecule, or to a conserved amino acid of the β-CASP region (possibly D165).
Mentions: The catalytic residues that define the two Zn(II)-binding pockets of class B β-Lacts concord to a highly conserved consensus signature: [HxHxD]…[H]…[C]…[H] (14). As most of these residues are conserved in Artemis (Fig. 1 B), we analyzed their role in Artemis function through in vitro mutagenesis. All engineered mutants were normally expressed and retained the capacity to interact with DNA-PKcs as judged by coimmunoprecipitation assay (Fig. 4 A). One representative experiment (Fig. 4 B) demonstrates that the basal V(D)J recombination activity in GUETEL/RSS cells (0.12%) is fully complemented by both Artemis (0.99%) or GST-Artemis (1.25%) expression constructs despite the previously reported reduced activity of a GST-Artemis fusion protein in vitro (10). GST-Artemis alone did not induce V(D)J recombination (0.01%) as expected. The integrated results of four experiments is shown in Fig. 4 C. Based on sequence analysis, the H33/H35/H115 triad in Artemis was predicted to correspond to the ligands of the first zinc (Zn1) of the binuclear Zn(II) center (Fig. 4 D). Accordingly, replacement of these residues with alanine strongly compromises the Artemis activity in all three mutants (Fig. 4 C). As a control, the mutant H151A, which affects a nonconserved His residue outside of the putative catalytic site, does not alter Artemis function. In the structure of class B enzymes as shown for Bacillus cereus in Fig. 4 D, the second zinc (Zn2) is coordinated by an aspartic acid, a histidine and a cysteine, and by two water molecules. The Asp residue is conserved in Artemis and its replacement by alanine (D37A) abolishes Artemis' activity. The Cys (Cys168 in B. cereus) is conserved in class B β-Lacts except for L1 enzyme of Stenotrophomonas maltophilia where it is replaced by a Ser at position 185, which is not a ligand of the zinc ion (14, 19). Instead, His89 provides the fifth Zn2 ligation in the L1 structure, next to the Asp88 residue in motif II (Figs. 1 B and 4 D). The Cys residue is not present in Artemis either, where Asp136 replaces it. Although this residue could substitute for the Cys in Zinc binding, it is interesting to note that Artemis also contains a His at position 38 in motif II that could participate in the Zn(II) coordination (Figs. 1 B and 4 D). To discriminate between these two possibilities, we analyzed the function of Artemis carrying alanine mutations at these two positions. Although H38A mutant is fully active, D136A has lost most of the catalytic activity (Fig. 4 C). Therefore, this result suggests that the putative second Zn(II) center of the Artemis catalytic site would adopt a structure similar to that of the B. cereus enzyme with Asp136 substituting the conserved Cys (Figs. 1 B and 4 D).

Bottom Line: Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates.Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity.The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

View Article: PubMed Central - PubMed

Affiliation: Développement Normal et Pathologique de Système Immunitaire, INSERM U429, Hôpital Necker Enfants Malades, 75015 Paris, France.

ABSTRACT
The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-beta-lactamase (beta-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the beta-Lact homology domain, to which is appended (b) the beta-CASP region, specific of members of the beta-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the beta-Lact and the beta-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the beta-Lact/beta-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B beta-Lact enzymes hydrolyzing beta-lactam compounds.

Show MeSH