Structural and biochemical studies on the chromo-barrel domain of male specific lethal 3 (MSL3) reveal a binding preference for mono- or dimethyllysine 20 on histone H4

Abstract

We have determined the human male specific lethal 3 (hMSL3) chromo-barrel domain structure by x-ray crystallography to a resolution of 2.5 Å (r = 0.226, R(free) = 0.270). hMSL3 contains a canonical methyllysine binding pocket made up of residues Tyr-31, Phe-56, Trp-59, and Trp-63. A six-residue insertion between strands β(1) and β(2) of the hMSL3 chromo-barrel domain directs the side chain of Glu-21 into the methyllysine binding pocket where it hydrogen bonds to the NH group of a bound cyclohexylamino ethanesulfonate buffer molecule, likely mimicking interactions with a histone tail dimethyllysine residue. In vitro binding studies revealed that both the human and Drosophila MSL3 chromo-barrel domains bind preferentially to peptides representing the mono or dimethyl isoform of lysine 20 on the histone H4 N-terminal tail (H4K20Me(1) or H4K20Me(2)). Mutation of Tyr-31 to Ala in the hMSL3 methyllysine-binding cage resulted in weaker in vitro binding to H4K20Me(1). The same mutation in the msl3 gene compromised male survival in Drosophila. Combined mutation of Glu-21 and Pro-22 to Ala in hMSL3 resulted in slightly weaker in vitro binding to H4K20Me(1), but the corresponding msl3 mutation had no effect on male survival in Drosophila. We propose MSL3 plays an important role in targeting the male specific lethal complex to chromatin in both humans and flies by binding to H4K20Me(1). Binding studies on the related dMRG15 chromo-barrel domain revealed that MRG15 prefers binding to H4K20Me(3).

FIGURE 1.

Tertiary structure of human MSL3 chromo-barrel domain. A, multiple sequence alignment of MSL3 and MRG15 CBD sequences from higher eukaryotes. Strictly conserved residues are shaded red, highly conserved residues yellow. A six-residue insertion specific to MSL3 is shaded blue, whereas a histidine residue found in the methyllysine binding cage of MRG15 is shaded green. Secondary structure assignments were derived from x-ray structures of hMSL3 (this work) and hMRG15 CBDs (PDB 2F5K) (41). The locations of the β1-β2 and β3-β4 loops making up the methyllysine binding pocket are marked with light green and magenta lines, respectively, above the sequences. Aromatic cage residues of the methyllysine binding pocket are marked with purple stars. The N-terminal 12 residues (MGEVKPAKVENY) of dMRG15 are not shown for clarity. B, ribbon diagram of hMSL3 residues 5–93 (subunit A in the crystal). The MSL3-specific loop between strands β1 and β2 is colored light green. Amino acid side chains associated with the presumed methyllysine binding pocket and a bound CHES buffer molecule are shown as stick models. C, qualitative electrostatic surface rendering of the hMSL3CBD (red negatively charged; blue positively charged, see “Experimental Procedures”) and bound CHES and sulfate anions are shown as stick representations. The hMSL3 CBD is rotated ∼90 degrees toward the viewer relative to B, looking directly into the methyllysine binding pocket.

FIGURE 2.

Comparison of methyllysine binding pockets in hMSL3, hMRG15, and h53BP1. A, methyllysine binding pocket in the hMSL3 CBD depicting the bound CHES buffer molecule (green carbons), colored as described in the legend to Fig. 1B. B, methyllysine binding pocket in the 53BP1 tandem tudor domain in blue (53) showing the bound H4K20Me₂ peptide with carbons colored light green. Only the first tudor domain is shown. C, methyllysine binding pocket in the hMRG15 CBD drawn in magenta with side chain carbons in light pink (41). Each pocket is drawn in an identical orientation, showing similar structural elements. Hydrogen bonds are drawn as purple dotted lines. Residues are labeled according to the text and the published structures. D, a potential peptide interaction surface on the hMSL3 CBD, the view is rotated ∼180° about the vertical axis relative to Fig. 1B. The hMSL3 CBD is rendered as a semi-transparent electrostatic surface (red negatively charged, blue positively charged) overlaid onto a ribbon diagram of the molecule. The bound CHES molecule (blue carbons) and the sulfate anion near His⁵⁵ are shown as stick representations. Residues mentioned in the text are labeled accordingly.

FIGURE 3.

Binding affinity of the human MSL3 chromo-barrel domain for the indicated methyllysine containing histone tail peptides. A, surface plasmon resonance steady state equilibrium binding for methylated histone tail peptides over the hMSL3 chromo-barrel domain (in response units normalized to maximum theoretical occupancy of ligand) in 250 mm NaCl, 3 mm EDTA, 100 mm HEPES pH 7.5. Individual data points and fitted curves based on the calculated K_d values are shown for each peptide series. B, semilog plot of data presented in A. The legend applies to both A and B. C, binding data for hMSL3 CBD with H4K20Me₁ and H4K20Me₃ in 150 mm NaCl buffer. D, binding data for WT hMSL3, the Glu²¹-Pro²²-Ala and Y31A hMSL3 CBD mutants with H4K20Me₁ in 150 mm NaCl running buffer.

FIGURE 4.

Binding affinity of the D. melanogaster MSL3 and MRG15 chromo-barrel domains for methyllysine containing histone tail peptides. A, surface plasmon resonance steady state equilibrium binding (in response units normalized to maximum theoretical occupancy of ligand) for the D. melanogaster CBD and histone tail peptides in 250 mm NaCl, 3 mm EDTA, 100 mm HEPES pH 7.5. Individual data points and fitted curves based on the calculated K_d values are shown for each peptide series. B, as in part A, but 150 mm NaCl and only H4K20Me₁ and H4K20Me₃. C, SPR binding of the dMRG15 CBD to H4K20Me₃, H4K20Me₂, and H3K36Me₂ in 150 mm NaCl running buffer.

FIGURE 5.

An electropositive surface on the hMSL3 CBD binds sulfate anions. A, the hMSL3 CBD is rendered as a opaque qualitative electrostatic surface (red negatively charged, blue positively charged, see “Experimental Procedures”). Sulfate anions bound at Lys¹⁰-Asn⁷⁹, His⁵⁵, and Arg²⁸ are shown as stick models (sulfur, yellow; oxygen, red). Green arrows point to regions that may interact with nucleic acids. B, ball and stick representation of two sulfate anions bound by conserved residues near Arg²⁸ and Lys¹⁰ on the surface represented in A. Backbone trace is colored as described in the legend to Fig. 1B.