Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex - PubMed (original) (raw)

Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex

Alexander J Ruthenburg et al. Nat Struct Mol Biol. 2006 Aug.

Abstract

WDR5 is a core component of SET1-family complexes that achieve transcriptional activation via methylation of histone H3 on Nzeta of Lys4 (H3K4). The role of WDR5 in the MLL1 complex has recently been described as specific recognition of dimethyl-K4 in the context of a histone H3 amino terminus; WDR5 is essential for vertebrate development, Hox gene activation and global H3K4 trimethylation. We report the high-resolution X-ray structures of WDR5 in the unliganded form and complexed with histone H3 peptides having unmodified and mono-, di- and trimethylated K4, which together provide the first comprehensive analysis of methylated histone recognition by the ubiquitous WD40-repeat fold. Contrary to predictions, the structures reveal that WDR5 does not read out the methylation state of K4 directly, but instead serves to present the K4 side chain for further methylation by SET1-family complexes.

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Figures

Figure 1

Figure 1

The overall structure of WDR5 complexes with histone H3. (a) The set of possible methylation states of H3K4. In general, nucleosomes di- and trimethylated at K4 of H3 are associated with transcriptional activation. (b) The overall structure of WDR5 in complex with H3K4me2. WDR5 is a canonical seven-bladed β-propeller (red); the histone H3 peptide binds across the top face of the protein (green, with amino (N′) and carboxyl (C′) termini and R2 labeled). (c) Electrostatic surface of WDR5 contoured from −8 to +8 kT using ABPS shows a series of acidic patches on the upper face of WDR5 (arrows 1–4), whereas the remainder of the protein is highly positively charged. H3K4me2 peptide is shown in green; potential aromatic cage residues are situated around arrow 5. Remaining arrows indicate patches of positive electrostatic potential discussed in the text. Cutaway view of this surface along the pseudo seven-fold symmetry axis (top) demonstrates how deeply buried R2 is in the cavity of the β-propeller fold's torus.

Figure 2

Figure 2

Bound histone H3 peptide electron density and conformation. (a) Stereo view of the H3K4me2 peptide model in a simulated annealing omit map contoured at 1.0 σ about the peptide, calculated from the protein model and the 1.5-Å data set (form I). (b) Main chain superposition of all of the WDR5-bound peptides. Nitrogen atoms are colored in blue, oxygen atoms in red, and carbon atoms are colored differently for each peptide as indicated in key. Points of conformational divergence are localized to the K4 and Q5 side chains.

Figure 3

Figure 3

Peptide recognition by WDR5 and conformational changes upon peptide binding. (a) The peptide is recognized by an elaborate series of direct and indirect contacts. Orientation of the peptide–WDR5 complex is the same as in the lower panel of Figure 1c. The majority of direct contacts from WDR5 are made to the N terminus and the first three residues. These residues adopt an approximately helical main chain conformation, with one hydrogen bond between the A1 and K4 backbone. Water-mediated contacts are important in recognition of the C-terminal residues of the peptide, as all waters shown (red spheres) are conserved among the peptide-bound structures. Tyr191 apparently acts as a central platform in this peptide-bound water network. (b) Phe133 and Phe263 form an aromatic sandwich about the R2 guanidinium moiety, equatorially flanked by a number of backbone carbonyl–mediated hydrogen bonds. These tight hydrogen bonds are thought to impart specificity for arginine over dimethyllysine, particularly the one from Nε of R2 to the Ser91 carbonyl. (c) Apparent coordinated movement of Phe133 and Phe149 to form the top of the aromatic sandwich recognition element when peptide is bound. The relevant apostructure side chains are depicted in gray and a representative liganded structure (H3K4me2 complex I) is in crimson. (d) Retraction of the loop bearing Lys259 causes a reorganization of the residues lining the central cavity, which permits tight R2 coordination. Coloring is as in c. This movement may be driven by a steric clash between this lysine and the incoming peptide Q5 side chain.

Figure 4

Figure 4

Peptide binding by WDR5. (a) Peptide injections over immobilized WDR5-biotin. Reference and blank traces were subtracted and traces from each functionalized flow cell (three traces per concentration) were averaged for ease of viewing. Injection concentrations of peptides (residues 1–14): H3 unmodified and H3K4me3, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0, 10.0 and 12.0 μM; H3K4me1, 1.0, 2.0, 4.0, 6.0, 10.0 and 12.0 μM; H3K4me2, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1.0, 2.0, 4.0 and 5.0 μM. The association/injection is from 0 to 200 s and the dissociation/buffer flow is from 200 to 460 s. The more gradual association and dissociation of H3K4me2 indicates both a slower on and slower off rate than those of the other peptides. Similar experiments with H31–9-derived peptide had slightly weaker binding but recapitulated the methyl-form binding trend shown. (b) Equilibrium _K_d analysis. Equilibrium _K_ds were determined by fitting response saturation fractions calculated by the Langmuir binding isotherm (see Methods). H3 unmodified, _K_d 3.3 ± 0.2 μM; H3K4me1, _K_d = 8.7 ± 0.3 μM; H3K4me2, _K_d = 1.02 ± 0.05 μM; H3K4me, _K_d = 7.8 ± 0.2 μM. (c) Single-protein injections of wild-type WDR5 and mutants, all at 750 nM, over immobilized H31–20-biotin peptides. Reference and blank traces were subtracted. Consistent with the importance of interactions with the N terminus of the H3 peptide, mutation of Asp107 to alanine caused the greatest decrease in peptide binding among all the mutant proteins examined. Mutation of Tyr131 to alanine leads to slightly enhanced binding of the H3 peptide, suggesting that even a methyl group is slightly larger than optimal for the A1-binding pocket. By contrast, the F149A mutant showed greatly decreased H3 peptide binding, perhaps owing to Phe149's additional role in stabilizing the R2-binding pocket.

Figure 5

Figure 5

Differences in K4 conformation in the different methylation states. (a–d) The crystal-packing interface relevant to K4 conformation is depicted for the H3K4me3 complex (_C_2 space group, a), H3K4me2 complex I (_C_2, b), H3K4me1 complex (_C_2, c) and unmodified H3 complex (_P_21, d). Peptides are colored as in Figure 2b; the principal WDR5 protomer is red; and the symmetry-related protomer at the peptide interface is gray. Note the rotation about χ3 moving from the tri- and dimethylated species to the monomethylated and unmodified species. In H3K4me2 complex I, the distances between the ζ-methyl carbons and the Glu322 carboxylate Oδ1 are 3.27 Å and 3.37 Å for the closest methyl group in each of the two complexes per asymmetric unit, whereas these distances are 3.83 Å and 3.87 Å for the more distant methyl group. For comparison, the previously reported shorter distances for these measurements were 3.15 Å and 3.42 Å.

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