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AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

Gaetani M, Matafora V, Saare M, Spiliotopoulos D, Mollica L, Quilici G, Chignola F, Mannella V, Zucchelli C, Peterson P, Bachi A, Musco G - Nucleic Acids Res. (2012)

Bottom Line: In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation.Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level.Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular NMR Laboratory, Center of Translational Genomics and Bioinformatics, Dulbecco Telethon Institute c/o S. Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

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(A) Solution structure of AIRE-PHD2. Superposition of the best 20 structures. Zn2+ (gray spheres) binding residues, Cys446, conserved hydrophobic amino acids and L3 are represented in yellow, blue, pink and green, respectively. (B) Backbone dynamics of AIRE-PHD2. The black box indicates residues of L3 displaying heteronuclear NOEs <0.65 (dotted line). (C) Superposition of 1H 15N HSQC spectra of AIRE-PHD1–PHD2 tandem domain (black), of AIRE-PHD1 (red) and of AIRE-PHD2 (green). (D) 1D 1H NMR spectra of AIRE-PHD2 wild-type and of C446G mutant.
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gks933-F1: (A) Solution structure of AIRE-PHD2. Superposition of the best 20 structures. Zn2+ (gray spheres) binding residues, Cys446, conserved hydrophobic amino acids and L3 are represented in yellow, blue, pink and green, respectively. (B) Backbone dynamics of AIRE-PHD2. The black box indicates residues of L3 displaying heteronuclear NOEs <0.65 (dotted line). (C) Superposition of 1H 15N HSQC spectra of AIRE-PHD1–PHD2 tandem domain (black), of AIRE-PHD1 (red) and of AIRE-PHD2 (green). (D) 1D 1H NMR spectra of AIRE-PHD2 wild-type and of C446G mutant.

Mentions: We solved AIRE-PHD2 3D structure by multidimensional heteronuclear NMR spectroscopy (Figure 1A). The recombinant protein (residues Q425-E485) behaves as a monomer in solution, as assessed by the rotational correlation time (τc ∼ 5 ns), determined from 15N relaxation data. This is in agreement with the expected value for a folded 7-kDa protein. Residues R433-S476 adopt a well-defined tertiary structure with an RMSD of 0.77 Å for backbone atoms and have all residues in the allowed regions of the Ramachandran plot (Table 1). In contrast, the N- and C-terminal residues are disordered as indicated by the small number of NOEs and negative heteronuclear NOE (Figure 1B). AIRE-PHD2 adopts the typical PHD finger fold characterized by a short two-stranded anti-parallel β-sheet (residues L444-R445 and A452-A453) followed by an α-helical turn and a C-terminal α-helix (residues R473-S476) (Figure 1A). The two Zn2+ ions are coordinated in a cross-braced scheme by C432, C437, H454, C457 and C446, C449, C472, C475, respectively. The domain is further stabilized by a network of conserved hydrophobic interactions composed by V443, L444, A452, F453, W455, F459, L470 (Figure 1A). Overall, the variable loops L1 (G338-R445) and L2 (A450-R456) are well defined, whereas L3 (H458-R471) is less ordered, in agreement with the paucity of inter residual NOEs in this region (Figure 1A and Supplementary Figure S1A). Consistently, residues in L3 show a reduction of the heteronuclear NOE intensities, thus indicating internal motions on a rapid time scale (pico- to nanosecond range) (Figure 1B). We observed similar dynamics in the corresponding loop in AIRE-PHD1 (19). Structural comparison performed with DALI server reveals high structural homology with AIRE-PHD1, with a Z-score of 3.9 and a RMSD of 1.6 Å over the Cα atoms of equivalent residues (Supplementary Figure S1C). In agreement with previous homology modeling studies (39), the analysis of the electrostatic surface potential revealed a large positively charged surface which is not suited for favorable interactions with the positively charged histone tails (Supplementary Figure S1B), indicating that the two PHD fingers—though structurally very similar—strongly diverge in function. Importantly, when linked in the context of the double domain (residues N295–E485), the two domains maintained their folds, behaved as structurally independent and non-interacting domains, as assessed from the 1H–15N HSQC spectrum of the tandem PHD1–PHD2 domain, which was essentially the sum of the 15N HSQC spectra of the single PHD domains (Figure 1C and Supplementary Figure S2A). Additional cross peaks, which belong to the linker region, were clustered in the middle of AIRE-PHD1–PHD2 spectrum, implying that the linker is largely unstructured. T1, T2 and {1H}15 N heteronuclear NOE experiments performed on the double domain construct show that the correlation time of the two PHD fingers within the tandem is similar (5.8 ± 0.2 ns; 6.2 ± 0.4 ns, for the first and second PHD finger, respectively) to the correlation time of the single domains (τc_PHD1 = 5.0 ± 0.4 ns; τc_PHD2 = 5.7 ± 0.5 ns) and the R2/R1 ratio follows the same trend as for the isolated domains (Supplementary Figure S2B). This indicates that the two PHD finger domains within the context of the tandem display independent overall tumbling and that they are not connected through tight contacts or interactions between each other, as the presence of a defined domain arrangement would lead to a change in the tensor components of the rotational diffusion tensor and to more native-like dynamics with significantly larger overall correlation time (overall τc_PHD1–PHD2 = 6.05 ± 0.4 ns AIRE). The {1H}15 N heteronuclear NOE values of both AIRE-PHD1 and AIRE-PHD2 in the tandem construct were similar to the values observed in the isolated domain (Supplementary Figure S2C), as expected, since the domains maintain their fold in the tandem construct and have therefore similar picosecond–nanosecond dynamics. Taken together, these data indicate that the two domains do not interact with each other and tumble independently in solution. Overall these results are in line with previous NMR studies in which titrations using the single domains did not highlight any intermolecular interactions (40). Finally, structural comparison with members of the RING finger family shows poor structural alignment (Supplementary Figure S1D and E), indicating structural differences between these two classes of zinc-binding domains.Figure 1.


AIRE-PHD fingers are structural hubs to maintain the integrity of chromatin-associated interactome.

Gaetani M, Matafora V, Saare M, Spiliotopoulos D, Mollica L, Quilici G, Chignola F, Mannella V, Zucchelli C, Peterson P, Bachi A, Musco G - Nucleic Acids Res. (2012)

(A) Solution structure of AIRE-PHD2. Superposition of the best 20 structures. Zn2+ (gray spheres) binding residues, Cys446, conserved hydrophobic amino acids and L3 are represented in yellow, blue, pink and green, respectively. (B) Backbone dynamics of AIRE-PHD2. The black box indicates residues of L3 displaying heteronuclear NOEs <0.65 (dotted line). (C) Superposition of 1H 15N HSQC spectra of AIRE-PHD1–PHD2 tandem domain (black), of AIRE-PHD1 (red) and of AIRE-PHD2 (green). (D) 1D 1H NMR spectra of AIRE-PHD2 wild-type and of C446G mutant.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526288&req=5

gks933-F1: (A) Solution structure of AIRE-PHD2. Superposition of the best 20 structures. Zn2+ (gray spheres) binding residues, Cys446, conserved hydrophobic amino acids and L3 are represented in yellow, blue, pink and green, respectively. (B) Backbone dynamics of AIRE-PHD2. The black box indicates residues of L3 displaying heteronuclear NOEs <0.65 (dotted line). (C) Superposition of 1H 15N HSQC spectra of AIRE-PHD1–PHD2 tandem domain (black), of AIRE-PHD1 (red) and of AIRE-PHD2 (green). (D) 1D 1H NMR spectra of AIRE-PHD2 wild-type and of C446G mutant.
Mentions: We solved AIRE-PHD2 3D structure by multidimensional heteronuclear NMR spectroscopy (Figure 1A). The recombinant protein (residues Q425-E485) behaves as a monomer in solution, as assessed by the rotational correlation time (τc ∼ 5 ns), determined from 15N relaxation data. This is in agreement with the expected value for a folded 7-kDa protein. Residues R433-S476 adopt a well-defined tertiary structure with an RMSD of 0.77 Å for backbone atoms and have all residues in the allowed regions of the Ramachandran plot (Table 1). In contrast, the N- and C-terminal residues are disordered as indicated by the small number of NOEs and negative heteronuclear NOE (Figure 1B). AIRE-PHD2 adopts the typical PHD finger fold characterized by a short two-stranded anti-parallel β-sheet (residues L444-R445 and A452-A453) followed by an α-helical turn and a C-terminal α-helix (residues R473-S476) (Figure 1A). The two Zn2+ ions are coordinated in a cross-braced scheme by C432, C437, H454, C457 and C446, C449, C472, C475, respectively. The domain is further stabilized by a network of conserved hydrophobic interactions composed by V443, L444, A452, F453, W455, F459, L470 (Figure 1A). Overall, the variable loops L1 (G338-R445) and L2 (A450-R456) are well defined, whereas L3 (H458-R471) is less ordered, in agreement with the paucity of inter residual NOEs in this region (Figure 1A and Supplementary Figure S1A). Consistently, residues in L3 show a reduction of the heteronuclear NOE intensities, thus indicating internal motions on a rapid time scale (pico- to nanosecond range) (Figure 1B). We observed similar dynamics in the corresponding loop in AIRE-PHD1 (19). Structural comparison performed with DALI server reveals high structural homology with AIRE-PHD1, with a Z-score of 3.9 and a RMSD of 1.6 Å over the Cα atoms of equivalent residues (Supplementary Figure S1C). In agreement with previous homology modeling studies (39), the analysis of the electrostatic surface potential revealed a large positively charged surface which is not suited for favorable interactions with the positively charged histone tails (Supplementary Figure S1B), indicating that the two PHD fingers—though structurally very similar—strongly diverge in function. Importantly, when linked in the context of the double domain (residues N295–E485), the two domains maintained their folds, behaved as structurally independent and non-interacting domains, as assessed from the 1H–15N HSQC spectrum of the tandem PHD1–PHD2 domain, which was essentially the sum of the 15N HSQC spectra of the single PHD domains (Figure 1C and Supplementary Figure S2A). Additional cross peaks, which belong to the linker region, were clustered in the middle of AIRE-PHD1–PHD2 spectrum, implying that the linker is largely unstructured. T1, T2 and {1H}15 N heteronuclear NOE experiments performed on the double domain construct show that the correlation time of the two PHD fingers within the tandem is similar (5.8 ± 0.2 ns; 6.2 ± 0.4 ns, for the first and second PHD finger, respectively) to the correlation time of the single domains (τc_PHD1 = 5.0 ± 0.4 ns; τc_PHD2 = 5.7 ± 0.5 ns) and the R2/R1 ratio follows the same trend as for the isolated domains (Supplementary Figure S2B). This indicates that the two PHD finger domains within the context of the tandem display independent overall tumbling and that they are not connected through tight contacts or interactions between each other, as the presence of a defined domain arrangement would lead to a change in the tensor components of the rotational diffusion tensor and to more native-like dynamics with significantly larger overall correlation time (overall τc_PHD1–PHD2 = 6.05 ± 0.4 ns AIRE). The {1H}15 N heteronuclear NOE values of both AIRE-PHD1 and AIRE-PHD2 in the tandem construct were similar to the values observed in the isolated domain (Supplementary Figure S2C), as expected, since the domains maintain their fold in the tandem construct and have therefore similar picosecond–nanosecond dynamics. Taken together, these data indicate that the two domains do not interact with each other and tumble independently in solution. Overall these results are in line with previous NMR studies in which titrations using the single domains did not highlight any intermolecular interactions (40). Finally, structural comparison with members of the RING finger family shows poor structural alignment (Supplementary Figure S1D and E), indicating structural differences between these two classes of zinc-binding domains.Figure 1.

Bottom Line: In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation.Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level.Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

View Article: PubMed Central - PubMed

Affiliation: Biomolecular NMR Laboratory, Center of Translational Genomics and Bioinformatics, Dulbecco Telethon Institute c/o S. Raffaele Scientific Institute, Milano, Italy.

ABSTRACT
Mutations in autoimmune regulator (AIRE) gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of peripheral-tissue antigens to mediate deletional tolerance, thereby preventing self-reactivity. AIRE contains two plant homeodomains (PHDs) which are sites of pathological mutations. AIRE-PHD fingers are important for AIRE transcriptional activity and presumably play a crucial role in the formation of multimeric protein complexes at chromatin level which ultimately control immunological tolerance. As a step forward the understanding of AIRE-PHD fingers in normal and pathological conditions, we investigated their structure and used a proteomic SILAC approach to assess the impact of patient mutations targeting AIRE-PHD fingers. Importantly, both AIRE-PHD fingers are structurally independent and mutually non-interacting domains. In contrast to D297A and V301M on AIRE-PHD1, the C446G mutation on AIRE-PHD2 destroys the structural fold, thus causing aberrant AIRE localization and reduction of AIRE target genes activation. Moreover, mutations targeting AIRE-PHD1 affect the formation of a multimeric protein complex at chromatin level. Overall our results reveal the importance of AIRE-PHD domains in the interaction with chromatin-associated nuclear partners and gene regulation confirming the role of PHD fingers as versatile protein interaction hubs for multiple binding events.

Show MeSH
Related in: MedlinePlus