Niedzialkowska E et al. (2012), Molecular basis for phosphospecific recognition...

XB-ART-44894

Mol Biol Cell 2012 Apr 01;238:1457-66. doi: 10.1091/mbc.E11-11-0904.

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Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres.

Niedzialkowska E, Wang F, Porebski PJ, Minor W, Higgins JM, Stukenberg PT.

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Survivin, a subunit of the chromosome passenger complex (CPC), binds the N-terminal tail of histone H3, which is phosphorylated on T3 by Haspin kinase, and localizes the complex to the inner centromeres. We used x-ray crystallography to determine the residues of Survivin that are important in binding phosphomodified histone H3. Mutation of amino acids that interact with the histone N-terminus lowered in vitro tail binding affinity and reduced CPC recruitment to the inner centromere in cells, validating our solved structures. Phylogenetic analysis shows that nonmammalian vertebrates have two Survivin paralogues, which we name class A and B. A distinguishing feature of these paralogues is an H-to-R change in an amino acid that interacts with the histone T3 phosphate. The binding to histone tails of the human class A paralogue, which has a histidine at this position, is sensitive to changes around physiological pH, whereas Xenopus Survivin class B is less so. Our data demonstrate that Survivin paralogues have different characteristics of phosphospecific binding to threonine-3 of histone H3, providing new insight into the biology of the inner centromere.

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Species referenced: Xenopus
Genes referenced: aurkb birc5l haspin myc

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	FIGURE 1:. Crystal structure of human Survivin with the N-terminal tail of histone H3 phosphorylated on T3. (A) Two orientations of Survivin rotated 90° relative to each other. Survivin is shown in green, and its surface is marked in gray. The peptide molecule is represented with red sticks. Insets show interactions between H3T3ph and Survivin. Inset shows the hydrogen bond network. H3T3ph carbon, nitrogen, oxygen, and phosphorus atoms are shown in dark red, blue, light red, and orange, respectively. Survivin carbon, nitrogen, and oxygen atoms of peptide-binding residues are shown as green, blue, and light red sticks, respectively. Water molecules are presented as light red spheres. Dashes depict probable hydrogen bonds. Marked residues are important in peptide binding. (B) Electrostatic potential on the Survivin surface near the peptide-binding groove. Negatively charged amino acids are marked in red, positively charged ones in blue. (C) Hydrophobicity level of binding pocket for H3T3ph mapped on the Survivin surface. Progressive color change from green to red indicates changes in amino acid hydrophobicity from hydrophilic (arginine [Arg]) to hydrophobic (isoleucine [Ile]).
	FIGURE 2:. Survivin interacts with the unmodified H3 tail in similar way as with H3T3ph. (A) Superposition of the crystal structure of wild-type Survivin with H3T3ph(1-4) (red lines) on the structure of wild-type Survivin (gray; cartoon representation) with H3(1-12) peptide (green in stick representation). For clarity, only D71, D76, H80, E65, and both peptides are shown in line or stick representation. Figures are in stereo representation. (B) Superposition of the crystal structure of wild-type Survivin with H3T3ph(1-4) (red lines) on the structure of K62A Survivin (gray; cartoon representation) with H3(1-12) peptide (green in stick representation). For clarity only, D71, D76, H80, E65, and both peptides are shown in line or stick representation. T3 Oγ can form a weak hydrogen bond with the H80 side chain if present in the modeled orientation. Alternatively, the hydrophobic interactions between T3 Cγ and Survivin may be formed if T3 adopts a different orientation (not shown). Determination of the prevailing orientation of T3 was not possible due to the low quality of its corresponding electron density. Figures are in stereo representation.
	FIGURE 3:. Chromosomal passenger complex delocalization in prometaphase in cells expressing mutant Survivin-myc proteins and depleted of endogenous Survivin by siRNA treatment. (A) HeLa cells stably expressing vector alone or Survivin-myc containing the indicated mutations were transfected with siRNA as shown, synchronized by double thymidine block and then released into fresh medium for 12 h, followed by immunostaining with anti–myc epitope antibody (green), anti–Aurora B antibody (red), and ACA. Scale bar, 5 μm. (B) For cells treated as in A, Aurora B localization classified by immunofluorescence microscopy in ∼100 cells in each condition is shown on the left. Means ± SD (n = 3). Right, the ratio of centromere to chromosome arm fluorescence intensity for Aurora B and Survivin-myc. Means ± SD (n = 5). *p < 0.001, p < 0.01, *p < 0.05 compared with cells expressing Survivin-myc WT by one-way analysis of variance followed by Bonferroni's multiple comparison test. ND, not determined.
	FIGURE 4:. Comparison between class A and class B Survivin paralogues. (A) Evolutionary relationships between Survivin proteins. The cladogram of Survivin protein based on sequence alignment in B. The evolution history was deduced using the minimum evolution method. The cladogram is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the cladogram. Numbers show bootstrap values at the nodes. (B) Sequence alignment of Survivin BIR domains from selected organisms. Residues involved in zinc finger motif formation are marked in yellow. Residues H and R, which distinguish class A and B Survivin, are marked in red. Progressive dark shading indicates identity between 80 and 100%. Numbers on the left side of the alignment indicate the residue from which the alignment begins; numbers on the right are the last residues taken into alignment. Numbers in parentheses are the number of amino acids present in insertions that were removed from alignment. Numbers above the alignment indicate the residue numbering in hSurvivin. Letters from A to D mark helices, and black arrows numbered from 1 to 3 mark β-strands. Residues that form hydrogen bonds with H3 peptide are marked by blue rectangles. N-terminal and C-terminal variable regions and insertions were removed from alignment for clarity. (C) Superposition of the crystal structure of wild-type hSurvivin (gray) and homology model of xSurvivin-B (green carbon atoms). Mutated residues in Survivin (human H80 and Xenopus R89) and histone H3T3ph(1-4) residues are marked. Green dashes depict probable hydrogen bonds between the homology model of xSurvivin-B and H3T3ph(1-4) peptide.
	FIGURE 5:. pH dependence of histone-binding affinity and phosphospecificity of class A and class B Survivins. (A) Differences in affinity of Survivin class A and class B toward H3T3ph(1-12) and H3(1-12) peptides depending on pH changes. Survivin class A is represented by hSurvivin, and Survivin class B is represented by xSurvivin. (B) Differences in phosphospecificity between Survivin class A (Homo sapiens) and class B (X. laevis) within pH range 6.8–8.2. Phosphospecificity is displayed as a ratio of the Survivin affinity to H3T3ph peptide to the affinity to H3 peptide, (1/KdH3T3ph)/(1/KdH3).

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