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Evolving Catalytic Properties of the MLL Family SET Domain

Summary Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase.

The sequence alignment iillustrates the overall conservation of the SET domain, but additionally local areas of divergence that lead to the segregation into three subgroups are apparent.

S2: Related to Figure 2. Establishing reaction conditions for in vitro methyltransferase assays
A series of preliminary assays were performed in order to establish a set of methyltransferase reaction conditions, applicable to both MLL1 and MLL4 constructs, in order to compare the methyltransferase activity of recombinant SET domain constructs with peptide substrate ( Figure S2). The aim was to ensure that the selected conditions were in the linear range for both enzymes whilst obtaining a reasonable signal for MLL1. All methyltransferase measurements were made in triplicate and the standard deviation indicated by the error bars. The data was normalised to the methyltransferase activity for the wild-type protein in each experiment. For the kinetic analysis (Figure 2A and B), raw scintillation values (DPM) were converted to nmoles CH 3 / min / µmol enzyme, based on the assumption that 1DPM is equivalent to 3 x 10 -14 mmoles CH 3 . In order to obtain an MLL4 SET domain construct that yielded diffraction quality crystals a combinatorial domain hunting strategy was employed. For this a short 6xHis tag was placed at the C-terminus of the domain and Nterminal truncation constructs generated and assessed for expression and solubility ( Figure S3-1).
Figure S3-1. Sequence introduced at the N-and C-terminus of the tagged construct.
A well-behaved construct starting at residue His 5382 successfully yielded diffracting crystals, and the subsequent structure is described in the main manuscript. However, when tested in the methyltransferase assay this Cterminally tagged construct exhibited negligible activity even when compared to MLL1 ( Figure S3-2). An equivalent construct but expressed with a cleavable tag at the N-terminus, leaving the C-term native, had high enzyme activity, but despite strenuous efforts did not yield diffracting crystals. The biochemical experiments presented in this manuscript describing the activity of the MLL4 SET domain were performed with MLL4 (5308-5537), which was also expressed with an N-terminal tag. This construct is slightly longer and incorporates the conserved WIN motif known to be required for WDR5 binding, and had essentially the same activity as the MLL4 (5382-5537) construct ( Figure S3-2). What impact does the tag have on the structure, and can the compromised activity be explained by the structure?
The introduced tag consists of eight residues KGHHHHHH, with the initial Lys replacing the native MLL4 C-terminal Asn residue ( Figure S3-1). Using the MLL1 structure containing an intact native C-terminus as a reference, the protein would naturally terminate immediately following the conserved Cys 4 Zn cluster, and make no contact with either the peptide binding groove or cofactor binding site ( Figure S3-3). In the MLL4 structure, following the Cys 4 Zn cluster, the tag extension makes a turn at the penultimate Met5536 residue and passes over the SAH cofactor ( Figure S3-3). Following the initial four tag residues, KGHH, there is a dramatic drop-off in electron density ( Figure S3-4), and only these residues could be built into the model unambiguously. This implies that the rest of the tag is flexible. There are features in the density that could correspond to partial occupancy by the remaining histidine imidazole rings, indicated in Figure S3-4. Three of these features cluster around the SAH cofactor, whist a fourth density feature is adjacent to residues Trp5395 and Ile5427 in the SET-N region of the adjacent symmetry related molecule. The main chain path linking these features is not sufficiently clear to build.
The initial tag residues interact with several features on the SET domain. Firstly the Lys points towards the solvent filled cavity between the postSET and SET-I region, and the high b-factors of this side chain indicate that it is flexible. When overlapped with the MLL1 ternary structure, the tag Lys side chain could clash with the histone H3 Gln5 residue and the first tag His with the implied position of the histone H3 Ala1 ( Figure S3-3). The main chain amide of the Gly forms a hydrogen bond with the His5521 side chain of the postSET loop. The first tag His side chain also interacts with this postSET loop residue and with the side chain of the Glu5440 residue, which is located on the SET-I helix. It cannot formally be ruled out that this interaction may influence the relative position of SET-I observed in the in MLL4 structure compared to MLL1. However the inferred flexibility of the tag, from the relatively high B-factors poor electron density, would support the hypothesis that the position of the SET-I determines that of the tag.
The second tag His side chain is orientated towards the solvent, and makes a solvent mediated interaction with the Tyr5472 hydroxyl of the neighbouring molecule, which suggests that the tag has a role in the formation of the crystal lattice.
The main chain amide and carbonyl of the first tag His make hydrogen bonds to the cofactor SAH O3' and O2' atoms respectively. A further water mediated hydrogen bond is made with the SAH O3' atom and the tag Lys carbonyl. These, in addition with the density features that may arise from the less ordered tag residues surrounding the cofactor, may compromise cofactor product release. The inactivity of the MLL4-His tagged construct is therefore likely to be the combination of two features, the partial obstruction of the substrate binding grove and an extended set of interactions with the cofactor.

SAH Phe Ala
The SET-I region of MLL1 SET domain adopts an atypical position that creates an unusually 'open' active site that is less optimal for methyl transfer. The position of the SET-I in MLL4 is more conventional. The region of the SET-I that packs against the target lysine residue is the channel peptide. To enclose the active site, the channel tetrapeptide packs against the first strand of the SET-C region. Comparing the MLL1 and MLL4 structures reveals that this interaction is less favourable for MLL1.
(A). Electron density map (2Fo-Fc) of the MLL4 channel tetrapeptide and first SET-C strand showing how the SET-I phenylalanine interacts with the Set-C alanine and creates the active site.
(B). List of the equivalent Set-I and Set-C residues from a range of SET domains. Only the MLL1 SET domain has a sequence mismatch, phenylaanine to serine, that generates a less favourable packing interaction.