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Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8.

Wang C, Zhang Q, Hang T, Tao Y, Ma X, Wu M, Zhang X, Zang J - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed.When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift.Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, Collaborative Innovation Center of Chemistry for Life Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People's Republic of China.

ABSTRACT
The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66(176-C) complexed with Rpl8(204-224) in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

No MeSH data available.


Oligomerization of NO66 is required for substrate binding and catalysis. (a) GST pull-down of NO66176–C by different fragments of Rpl8 fused to GST. Asterisks indicate the band corresponding to NO66176–C. (b) A comparison of the efficiency of wild-type NO66 and mutants. The products of the hydroxylation reaction were detected by LC-MS/MS and the data are semi-quantitative. (c, d) Comparison of the binding of wild-type NO66176–C (red) and mutant protein M2 (black) to GST-Rpl8193–C (c) and αKG (d). The ITC method was used to analyze the binding affinities.
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fig1: Oligomerization of NO66 is required for substrate binding and catalysis. (a) GST pull-down of NO66176–C by different fragments of Rpl8 fused to GST. Asterisks indicate the band corresponding to NO66176–C. (b) A comparison of the efficiency of wild-type NO66 and mutants. The products of the hydroxylation reaction were detected by LC-MS/MS and the data are semi-quantitative. (c, d) Comparison of the binding of wild-type NO66176–C (red) and mutant protein M2 (black) to GST-Rpl8193–C (c) and αKG (d). The ITC method was used to analyze the binding affinities.

Mentions: NO66 was recently identified as a protein hydroxylase that is capable of catalyzing histidyl hydroxylation of Rpl8 (Ge et al., 2012 ▸). To confirm the activity of NO66, we evaluated the interaction between NO66 and Rpl8. We performed in vitro pull-down assays with several fragments of Rpl8 fused to GST. As expected, the C-terminal fragment of Rpl8 binds to NO66176–C (Fig. 1 ▸a). In addition, a short region of Rpl8 spanning amino-acid residues 193–224 is sufficient to interact with NO66176–C (Fig. 1 ▸a). Next, we examined the histidyl hydroxylation activity of NO66176–C by using a synthetic peptide containing 21 amino-acid residues of Rpl8 (204–224; referred to as Rpl8204–224) as the substrate. After incubation with NO66176–C, the molecular weight of Rpl8204–224 had a +16 Da shift, indicating the possible occurrence of hydroxylation (Supplementary Fig. S1a). Further analyses showed that His216 of Rpl8 is hydroxylated by NO66176–C (Supplementary Fig. S1b). Our activity assays confirmed that NO66 is a protein histidyl hydroxylase.


Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8.

Wang C, Zhang Q, Hang T, Tao Y, Ma X, Wu M, Zhang X, Zang J - Acta Crystallogr. D Biol. Crystallogr. (2015)

Oligomerization of NO66 is required for substrate binding and catalysis. (a) GST pull-down of NO66176–C by different fragments of Rpl8 fused to GST. Asterisks indicate the band corresponding to NO66176–C. (b) A comparison of the efficiency of wild-type NO66 and mutants. The products of the hydroxylation reaction were detected by LC-MS/MS and the data are semi-quantitative. (c, d) Comparison of the binding of wild-type NO66176–C (red) and mutant protein M2 (black) to GST-Rpl8193–C (c) and αKG (d). The ITC method was used to analyze the binding affinities.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556315&req=5

fig1: Oligomerization of NO66 is required for substrate binding and catalysis. (a) GST pull-down of NO66176–C by different fragments of Rpl8 fused to GST. Asterisks indicate the band corresponding to NO66176–C. (b) A comparison of the efficiency of wild-type NO66 and mutants. The products of the hydroxylation reaction were detected by LC-MS/MS and the data are semi-quantitative. (c, d) Comparison of the binding of wild-type NO66176–C (red) and mutant protein M2 (black) to GST-Rpl8193–C (c) and αKG (d). The ITC method was used to analyze the binding affinities.
Mentions: NO66 was recently identified as a protein hydroxylase that is capable of catalyzing histidyl hydroxylation of Rpl8 (Ge et al., 2012 ▸). To confirm the activity of NO66, we evaluated the interaction between NO66 and Rpl8. We performed in vitro pull-down assays with several fragments of Rpl8 fused to GST. As expected, the C-terminal fragment of Rpl8 binds to NO66176–C (Fig. 1 ▸a). In addition, a short region of Rpl8 spanning amino-acid residues 193–224 is sufficient to interact with NO66176–C (Fig. 1 ▸a). Next, we examined the histidyl hydroxylation activity of NO66176–C by using a synthetic peptide containing 21 amino-acid residues of Rpl8 (204–224; referred to as Rpl8204–224) as the substrate. After incubation with NO66176–C, the molecular weight of Rpl8204–224 had a +16 Da shift, indicating the possible occurrence of hydroxylation (Supplementary Fig. S1a). Further analyses showed that His216 of Rpl8 is hydroxylated by NO66176–C (Supplementary Fig. S1b). Our activity assays confirmed that NO66 is a protein histidyl hydroxylase.

Bottom Line: Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed.When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift.Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, Collaborative Innovation Center of Chemistry for Life Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People's Republic of China.

ABSTRACT
The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66(176-C) complexed with Rpl8(204-224) in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

No MeSH data available.