Volume 33, Issue 2, Pages 181-191 (January 2009) Structural Basis for the Requirement of Additional Factors for MLL1 SET Domain Activity and Recognition of Epigenetic Marks  Stacey M. Southall, Poon-Sheng Wong, Zain Odho, S. Mark Roe, Jon R. Wilson  Molecular Cell  Volume 33, Issue 2, Pages 181-191 (January 2009) DOI: 10.1016/j.molcel.2008.12.029 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Overall Structure of the MLL1 SET Domain (A) Schematic representation of the full-length MLL1 protein and the construct containing the SET domain used in structural studies (residues 3785–3969), indicating the subdomains referred to in the text. The N-flanking region is in pale blue, the SET-N in pale green, the SET-I in blue, the SET-C in bright green, and the postSET domain in orange. (B) Sequence of the MLL1 SET domain aligned with MLL1, Dim-5, Suv39h2, Set7/9, and PRSet7. The SET domain region secondary structure elements derived from the structure are indicated above the sequence. Identical residues are highlighted in red, conserved cysteines involved in Zn binding in orange, and active site residues in blue. (C) Two views of a cartoon representation of the MLL1 SET domain structure with a stick representation of the cofactor product AdoHcy (gray) and histone peptide substrate (purple). Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 The MLL1 Active Site and Activity (A) Residues which form the active site of MLL1 and help form the lysine binding channel. The electron density (2Fo-Fc) map, contoured to 1σ, is shown for these residues. (B) Activity assay of the MLL1 SET domain carried out in vitro with substrate peptides based on histone H3 either unmodified, or carrying K3K4me1 or H3K4me2 modifications for wild-type and site-directed mutants. Activity is represented as the mean of triplicate measurements, with error bars indicating one standard deviation. Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Ordering of the MLL1 Active Site (A) Overlay of the SET-I region and C-flanking region of MLL1 (gray), and Dim-5 (yellow, PDB; 1PEG). Structures are superposed on their cofactors and overlap on their C-flanking domains. (B) Superposition of active site residues in the SET domains of MLL1 and Dim-5 showing the displacement of the channel tetrapeptide in MLL1. (C) Reorientation of SET-I; surface representation of the MLL1 ternary complex on the left showing open configuration of the active site. On the right, the MLL1 Set-I domain (green) has been modeled to align with the position observed in Dim5. (D) Schematic representation of the MLL1 complex. (E) Methyltransferase activity of MLL1 in the presence of Ash2L-Dpy30 heterodimer Wdr5-RbBP5 heterodimer, Wdr5, RbBP5, and Ash2L-DPY30 + Wdr5-RbBP5. Activity is represented as the mean of triplicate measurements, with error bars indicating one standard deviation. Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Mutagenesis of the SET-I Helix (A) Representation of the SET-I helix showing the electrostatic surface potential, negative in red and positive in blue. Three pairs of residues are highlighted, which appear to form surface features. (B) Methyltransferase assay with unmodified histone peptide for wild-type and SET-I mutant proteins. Activity is represented as the mean of triplicate measurements, with error bars indicating one standard deviation. Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Substrate Binding (A) Details of the residues involved in binding of the substrate peptide. Water molecules are shown as red spheres, and hydrogen bonds are shown as gray dots. (B) Methyltransferase activity of the isolated MLL1 SET domain with substrate peptides carrying physiologically relevant posttranslational modifications. Activity is represented as the mean of triplicate measurements, with error bars indicating one standard deviation. Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 The C-Flanking Domain and Cofactor Binding (A) Representation of the Cys(4)Zn cage formed by three residues from the postSET domain and one from the SET-C region. Bond distances between the cysteine sulfur and Zn ion are indicated in black. (B) Surface view of the MLL1 SET domain showing the surface potential and illustrating the peptide-binding groove and cofactor-binding pocket. Areas of negative potential are in red, and areas of positive potential are in blue. (C) Details of cofactor binding. Residues involved in AdoHcy binding are shown in stick representation colored according to Figure 1A, and key hydrogen bonds are indicated by a red dotted line. Molecular Cell 2009 33, 181-191DOI: (10.1016/j.molcel.2008.12.029) Copyright © 2009 Elsevier Inc. Terms and Conditions