Fig. 2. Structure of the Runt domain–CBFβ heterodimeric complex. (A) A stereoscopic diagram of the C_α trace of the AML1 Runt domain, residues 54–178, bound to CBFβ residues 2–135, prepared with MOLSCRIPT (Kraulis, 1991). The numbering corresponds to the amino acid sequences of human AML1 (Miyoshi et al., 1991) and human CBFβ (Liu et al., 1993). (B) Ribbon diagram of a dimer of Runt domain–CBFβ heterodimers (two per asymmetric unit). Runt domain, cyan; CBFβ, magenta. The Runt domain β-strands are labelled βO to βG, consistent with the established immunoglobulin fold nomenclature and with Nagata et al. (1999), except for the extensions to strands βA and βG, which have been labelled βA′ and βG′, respectively. The β-strands of CBFβ are labelled β1–6, and the helices are numbered H1–5, consistent with previous nomenclature (Goger et al., 1999). Only one hydrogen bond corresponding to a short potential 3₁₀-helix was seen in the region corresponding to helix H4, which was therefore not represented. CBFβ residues 73–78 are disordered and are shown as a dashed line.

Fig. 4. The mode of interaction between the Runt domain and CBFβ. (A) Runt domain (cyan) and CBFβ (magenta) viewed perpendicular to the long axis of CBFβ. The concave surface of the Runt domain β-sheet, formed from strands βG, βF and βC, packs against the complementary convex strand β3 of CBFβ. (B) Two views of the Runt domain–CBFβ structure (i and ii), related by a 180° rotation about the vertical axis. The long axes of the CBFβ and Runt domain β-barrels are orthogonal to one another. (C) Electrostatic surface potential of the Runt domain--CBFβ heterodimer. The two views are related by a 145° rotation about the vertical axis. Positive areas are shaded blue; negative areas are shaded red. This figure was prepared using GRASP (Nicholls et al., 1991). (i) Positive surface. Labelled residues are mutated in cleidocranial dysplasia, familial platelet disorder and sporadic acute myeloid leukaemia. (ii) Negative surface. The evolutionarily conserved, negatively charged residues that are labelled are all located on the surface of CBFβ.

Fig. 6. The Runt domain complexed to CBFβ has a novel conformation. Least squares superposition of the C_α trace of the Runt domain crystal structure onto the most closely related NMR conformer [PDB code 1cmo, number 38, (Nagata et al., 1999)]. The analysis was performed for all the NMR conformers, and similar results were obtained. The NMR structure is shown in brown and the crystal structure is shown in cyan. The crystal and NMR structures differ in the relative positions of the βA′–βG′ sheet and the βF--G loop. The C-terminus is well defined in the crystal structure.

Fig. 1. B-factor distribution and crystal packing of Runt domain–CBFβ heterodimers. Six Runt domain subunits and four CBFβ subunits are packed in alternating layers in the crystal. One layer is composed of two dimers of heterodimers (F+E, G+H; B+A, C+D), and the second comprises a single Runt domain homodimer (Q+R). The subunits are represented as C_α traces, and are coloured according to temperature (B) factors. Colours are graded blue (≤30 Å²) through to red (≥70 Å²). Runt domain subunits are labelled A, C, E, G, Q and R; CBFβ subunits are labelled B, D, F and H. The view is down the b-axis with a, c and crystallographic 2₁ axes indicated.

Fig. 3. Amino acid sequence alignment of the Runt domain and CBFβ. (A) The amino acid sequence accession numbers for the Swiss-Prot and DDBJ/EMBL/GenBank databases are given in parentheses. The sequence information is derived from human AML1 (Q01196), residues 50–178; human AML2 (Q13761), residues 54–182; human AML3 (Q08775), residues 102–220; murine PEBP2αA (Q08775, D14636), residues 50–178; sea-urchin SpRunt-1 (Q26628), residues 57–185; frog Xaml1 (O73725), residues 50–178; fruit-fly Lozenge (Q24183), residues 278–406 and Runt (Q24709), residues 106–234; nematode Run (O01834), residues 10–138. The last line of the alignment shows the structural similarity of the Runt domain to murine STAT3β (P42227), residues 318–472, with manually introduced sequence gaps indicated. Identical residues are highlighted in red, conservatively substituted residues are highlighted in yellow. Sequences were aligned using CLUSTAL_W (Thompson et al., 1994) and manually adjusted. (B) Sequence alignment of human CBFβ (Q13951), residues 1–135, and the fruit-fly homologues Brother (Q24039) and Big Brother (Q24040). Residues 70–74 in CBFβ have no equivalent in Drosophila, and are indicated by dashed lines.

Fig. 5. Interaction surfaces within the (Runt domain–CBFβ)₂ complex. (A) Heterodimerization surface of CBFβ. Solvent-accessible surface is shown in purple; residues buried in the Runt domain surface are shown in cyan. All interface residues apart from Q67 and P100 are conserved (see also Figure 3). (B) Heterodimerization surface of the Runt domain. Solvent-accessible surface is shown in cyan; residues buried in the interface with CBFβ are shown in magenta. The Runt domain homodimer binding partner is shown in worm representation (cyan). (C) Homodimerization surface of the Runt domain. Solvent-accessible surface is shown in cyan; buried residues are shown in orange. The related heterodimeric CBFβ subunit is shown in worm form (magenta).

Fig. 7. Location of human disease mutations on the AML1–CBFβ structure. (A) A ribbon diagram of the Runt domain–CBFβ heterodimer in which residues mutated in cleidocranial dysplasia (CCD) are shown in yellow, familial platelet disorder (FPD) and acute myeloid leukaemia (AML) are shown in blue. Mutations common to CCD and FPD/AML are shown in green. The ribbon representation of the Runt domain is in cyan, and that of CBFβ is in magenta. (The AML-associated biallelic point mutations G138D, R135G, D171G are a personal communication from P.Fenaux.) (B) Model orienting the Runt domain–CBFβ heterodimer with respect to B-form DNA. The model was generated by least squares superposition of the co-ordinates of the STAT3β–DNA complex (PDB code, 1bg1) (Becker et al., 1998) onto the AML1 Runt domain. The amino acid residues mutated in human disease mutations are indicated as blue spheres. The Runt domain is shown in cyan, and CBFβ in magenta. The model is consistent with: the electrostatic surface potential of the heterodimer (Figure 4C); in vitro mutagenesis data (Lenny et al., 1995; Kagoshima et al., 1996; Osato et al., 1999); chemical footprinting analysis (Thornell et al., 1988; Melnikova et al., 1993); NOE data from NMR studies (Nagata et al., 1999); and the observations that CBFβ does not contact DNA directly, or extend the Runt domain footprint on DNA (Kamachi et al., 1990).