Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFbeta
2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês
10.1093/emboj/19.12.3004
ISSN1460-2075
Autores Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle15 June 2000free access Structural basis for the heterodimeric interaction between the acute leukaemia-associated transcription factors AML1 and CBFβ Alan J. Warren Corresponding Author Alan J. Warren MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 2XY UK Search for more papers by this author Jerónimo Bravo Jerónimo Bravo MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Roger L. Williams Roger L. Williams MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Terence H. Rabbitts Corresponding Author Terence H. Rabbitts MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Alan J. Warren Corresponding Author Alan J. Warren MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 2XY UK Search for more papers by this author Jerónimo Bravo Jerónimo Bravo MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Roger L. Williams Roger L. Williams MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Terence H. Rabbitts Corresponding Author Terence H. Rabbitts MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information Alan J. Warren 1,2, Jerónimo Bravo1, Roger L. Williams1 and Terence H. Rabbitts 1 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK 2Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Hills Road, Cambridge, CB2 2XY UK *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2000)19:3004-3015https://doi.org/10.1093/emboj/19.12.3004 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mutations in the genes encoding the interacting proteins AML1 and CBFβ are the most common genetic abnormalities in acute leukaemia, and congenital mutations in the related AML3 gene are associated with disorders of osteogenesis. Furthermore, the interaction of AML1 with CBFβ is essential for haematopoiesis. We report the 2.6 Å resolution crystal structure of the complex between the AML1 Runt domain and CBFβ, which represents a paradigm for the mode of interaction of this highly conserved family of transcription factors. The structure demonstrates that point mutations associated with cleidocranial dysplasia map to the conserved heterodimer interface, suggesting a role for CBFβ in osteogenesis, and reveals a potential protein interaction platform composed of conserved negatively charged residues on the surface of CBFβ. Introduction Leukaemias are characterized by the presence of recurrent chromosomal translocations (Rabbitts, 1994). The genes associated with these chromosomal breakpoints in acute leukaemias frequently encode transcription factors that play pivotal roles in normal development and in leukaemogenesis (Cleary, 1991; Rabbitts, 1991). The core binding factors (CBFs) are representative of this phenomenon. These heterodimeric transcription factors consist of a DNA-binding α-subunit, and a non-DNA-binding β-subunit (Ogawa et al., 1993b). Three mammalian genes encode the α-subunit: AML1/CBFA2/PEPBP2αB (herein called AML1), AML2/CBFA3/PEBP2αC and AML3/CBFA1/PEBP2αA/Osf2 (herein called AML3). All α-subunits share an evolutionarily conserved region of 128 amino acids known as the Runt domain, which mediates both DNA binding and heterodimerization to the β-subunit. The Drosophila gene runt, which is the founding member of the α-subunit family, is required for segmentation, sex determination and neurogenesis. Only one gene (CBFB) is known to encode the mammalian β-subunit, CBFβ, which associates with all three α-subunits (Ogawa et al., 1993a). However, two CBFB homologues, brother and big brother, have been identified in Drosophila (Golling et al., 1996). The AML1 gene encodes a 453 amino acid protein with an N-terminal transcriptional inhibitory domain (residues 1-49), the Runt domain (residues 50-177), and C-terminal transcriptional inhibition (residues 178-290) and activation (residues 291-453) domains (Ito, 1999). It was cloned from one of the most frequently acquired cytogenetic abnormalities in acute myeloid leukaemia (AML), the translocation t(8;21)(q22;q22), and was subsequently shown to be involved in the recurrent chromosomal translocation t(12;21)(p13;q22) associated with childhood acute lymphoblastic leukaemias, and the translocation t(3;21)(q26;q22) associated with therapy-related leukaemias and myelodysplasia (reviewed in Look, 1997). In all of these translocations, the AML1 Runt domain becomes fused with new protein domains encoded by exons from the partner chromosome, thereby retaining the ability to heterodimerize with the CBFβ protein. Additionally, nonsense, missense and frameshift mutations in the AML1 gene are associated with sporadic AML (Osato et al., 1999), and congenital mutations in AML1 have been described in individuals with the rare autosomal dominant disease, familial platelet disorder (FDP), in which there is a congenital predisposition to the development of AML (Song et al., 1999). Interestingly, these disease-associated mutations in the sporadic and congenital disorders are clustered within the Runt domain of AML1. In man, mutations in AML3 are associated with cleidocranial dysplasia (CCD), an autosomal dominant disorder of skeletal morphogenesis (Lee et al., 1997; Mundlos et al., 1997), and again, the majority of the point mutations associated with this disorder cluster within the Runt domain (Lee et al., 1997; Quack et al., 1999; Zhou et al., 1999). Aml3 is essential for osteoblast differentiation and bone development in the mouse (Komori et al., 1997; Otto et al., 1997). Thus, the conserved Runt domain of this family of transcription factors is a key target for disease-associated mutations in man. It is significant that the gene encoding CBFβ, the β-subunit of the core binding factors family, is also involved in chromosomal translocations in AML as a result of inv(16)(p13q22), t(16;16) and del(16)(q22), which fuse the N-terminal 165 amino acids of CBFβ in-frame with a C-terminal portion of the smooth muscle myosin heavy chain in 15% of AML (Liu et al., 1993). Thus, together, the heterodimeric CBF transcription factor genes AML1 and CBFB are the most frequently mutated genes in human acute leukaemia, accounting for 25% of AML and 20% of paediatric common B-cell acute lymphoblastic leukaemia (Look, 1997). AML1 binds as a monomer to the core DNA sequence TGT/cGGT, which is present in a number of different viral and cellular promoters and enhancers, as well as haematopoietic cell-specific genes (Rodan and Harada, 1997). The Runt domain binds to the major groove of DNA (Thornell et al., 1988), and dimerization with CBFβ enhances the DNA-binding activity of AML1 without itself contacting DNA (Kamachi et al., 1990). Binding of CBFβ to the Runt domain protects Cys81 from oxidation by diamide, but does not hinder access of much larger reducing molecules to this site (Akamatsu et al., 1997a). These data suggest that the Runt domain undergoes a conformational change on binding to CBFβ, which results in enhanced DNA binding and alters the susceptibility of Cys81 to oxidation. However, direct evidence for this has not been obtained. We have determined the structure of the Runt domain of AML1 bound to CBFβ to understand the mode of interaction between the two subunits, to investigate the mechanism whereby CBFβ enhances DNA binding by the Runt domain, and to understand the molecular consequences of physiologically relevant mutations. We describe the 2.6 Å resolution crystal structure of the human AML1-CBFβ complex, providing insights into the significance of human disease mutations associated with acute leukaemia and cleidocranial dysplasia. Results Formation of the Runt domain-CBFβ complex, crystallization and structure determination Fragments of the human AML1 and CBFβ proteins were co-expressed in Escherichia coli. The strategy of co-expression was crucial to obtaining a soluble functional heterodimeric complex without a requirement for mutagenesis or refolding. The expressed fragments of human AML1 (residues 50-183, out of 451), corresponding to the Runt domain, and CBFβ (residues 2-135, out of 182) form a stable complex in solution that is fully active in sequence-specific DNA binding as determined by electrophoretic mobility shift assays (data not shown). Two crystal forms of the complex were obtained, one with P61 symmetry and the second with P21 symmetry. The P21 symmetry crystals diffracted to higher resolution and were used in the structure determination. The structure was solved by the method of multiple anomalous dispersion (MAD) (Hendrickson et al., 1990), using isomorphous crystals produced from seleno-methionine (SeMet)-substituted protein. Crystallographic phases were determined using data sets collected at two wavelengths, measured at the ESRF, Grenoble, from crystals maintained at 100 K (Table I). Phases were extended to 2.6 Å resolution, and the resulting electron density map was of sufficient quality to build the initial model. The structure has been refined to a free R-factor of 29.4%, with no residues in disallowed regions of the Ramachandran plot (Table I). Table 1. Data collection, structure determination and refinement statistics Data collection and MIR phasing statistics Data set Resolution (Å) Observations/unique reflections Completeness(last shell) % Rmergea (last shell) SeMet λ1 3.2 78580/60509 88.9 (84.1) 0.047 (0.15) SeMet λ2 3.2 86114/63424 92.8 (85.4) 0.066 (0.25) Native 2.6 259515/71198 99.3 (99.2) 0.084 (0.28) Structure refinement statistics Resolution (Å) Protein atoms Waters Rcrystb Rfreeb (% data used) R.m.s.d. from idealityc Bonds Angles Dihedrals 25.0-2.6 9551 121 26.46 29.43 (3) 0.0072 1.3921 24.76 a Rmerge = ΣhklΣi|Ii(hkl) − |/ΣhklΣi Ii(hkl). b Rcryst and Rfree = Σ|Fobs − Fcalc|/ΣFobs; Rfree calculated with the percentage of the data shown in parentheses. c R.m.s.ds for bond angles and lengths in regard to Engh and Huber parameters. Overall structure of the Runt domain-CBFβ heterodimeric complex The asymmetric unit of the crystal contains six Runt domain subunits and four CBFβ subunits arranged as two dimers of Runt domain-CBFβ heterodimers [(Runt domain-CBFβ)2], and a Runt domain homodimer (Figure 1). The molecules pack in alternate layers of heterodimers and homodimers. The final atomic model is most complete for the heterodimer CD, which includes residues 54-177 of AML1, and residues 2-135 from the CBFβ subunit. Residues 52-53 and 178-183 of the Runt domain are disordered, as are residues 75-80 from CBFβ, and are not included in the model. One-hundred and twenty-one water molecules are also included. The Runt domain homodimer QR (Figure 1) has electron density at the interfaces where CBFβ binds in the heterodimers, but not sufficient to indicate an ordered CBFβ subunit. Attempts to refine with a model fitted into this density resulted in a higher free R-factor. This density may represent partial occupancy by CBFβ subunits. We have noted the formation of (Runt domain-CBFβ)2 dimers mediated by interactions between the Runt domain N-terminal residues in two different crystal forms (P21 and P61 symmetry) and in three independent examples in the asymmetric unit of the P21 crystal form. Gel filtration studies (data not shown) suggest that the formation of (Runt domain-CBFβ)2 dimers is not a consequence of crystal packing, but reflects homodimerization of the Runt domain-CBFβ complex in solution. Figure 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 Cα 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. Download figure Download PowerPoint The overall structure of the (Runt domain-CBFβ)2 dimer is shown in Figure 2. Although the fold of the individual subunits of the complex is consistent with recent NMR studies (Berardi et al., 1999; Goger et al., 1999; Huang et al., 1999; Nagata et al., 1999), specific regions of the Runt domain differ in conformation (discussed later). The Runt domain of AML1 forms a 12-stranded (10 antiparallel and two parallel strands) β-barrel that adopts an s-type immunoglobulin (Ig) fold (Bork et al., 1994). CBFβ is a mixed α/β structure, consisting of a partly open six-stranded β-barrel with α-helices packed against the top and bottom. Although structural comparisons with the DALI database show that the β-barrel component of CBFβ has overall structural similarity to a number of functionally unrelated proteins, it appears that the fold is distinct, as opposed to a possible relationship to the OB fold (Goger et al., 1999). As classified in the SCOP protein structure database (Murzin et al., 1995), the OB fold consists of a five-stranded β-barrel, with Greek key topology and a shear number of 8 or 10. CBFβ forms a partly opened six-stranded β-barrel with a unique combination of a meandering up and down topology of the β-strands and a shear number of 10. Consequently, this particular β-barrel structure can be classified as a novel fold from the distinct combination of topology and shear number. Figure 2.Structure of the Runt domain-CBFβ heterodimeric complex. (A) A stereoscopic diagram of the Cα 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. Download figure Download PowerPoint There are extensive heterodimeric contacts at the interface between the Runt domain and CBFβ subunits, and homodimeric contacts between the N-termini of the Runt domains, but no contacts between the CBFβ subunits. The temperature factors for the (Runt domain-CBFβ)2 structure (Figure 1) suggest that the Runt domain forms a relatively stable core, whereas the CBFβ subunit is more mobile. Figure 3 shows the secondary structure elements for the conserved Runt domain and CBFβ determined from our structure, aligned to the protein sequences of various family members. Figure 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. Download figure Download PowerPoint The root mean square deviations (r.m.s.ds) of the Cα backbone traces between different Runt domain subunits in the asymmetric unit range between 0.15 and 0.35 Å, and for the four CBFβ subunits, the values range between 0.16 and 0.36 Å. The overall dimensions of a single Runt domain-CBFβ heterodimer are ∼41 × 50 × 27 Å. The Runt domain and CBFβ interact along a large continuous curved interface (Figure 4A), and are oriented such that the long axes of the two β-barrel domains are orthogonal to one another (Figure 4B). When viewed from the perspective of Figure 4B, with the C-terminus of the Runt domain oriented downward, it is evident that CBFβ makes contact only with the upper part of the Runt β-barrel. CBFβ makes no direct contact with Runt domain loops βA′-B, βE′-F or the C-terminus. Residues within these loops have been shown biochemically to be essential for DNA binding (Kagoshima et al., 1996; Osato et al., 1999). The α-helices H5, H1 and H2 of CBFβ lie on the right lateral aspect of the upper half of the Runt β-barrel, and helix H3 lies on the upper left lateral aspect (Figure 4Bii). Our data differ from the proposed mode of interaction between the Runt domain and CBFβ, based on chemical shift analysis (Nagata et al., 1999). It was suggested that CBFβ is oriented in the heterodimer with helices H1, H2 and H5 up, and helix H3 down, placing CBFβ residues Gln74, Gln79 and Arg83 on the same face of the heterodimer as the proposed DNA-binding surface of the Runt domain (loops βA′-B and βE′-F and the C-terminus). In fact, the crystal structure demonstrates that the CBFβ is rotated by 90° relative to the previous proposal (Figure 4B), so that the evolutionarily non-conserved CBFβ loop β3-β4 (residues 68-93) makes no contribution either to the heterodimer interface or to the DNA binding surface of the molecule. Figure 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. Download figure Download PowerPoint The conformation of the C-terminus of the Runt domain (residues 169-177), which is essential for DNA binding, is clearly defined in the crystal structure. The C-terminus forms a loop that extends towards the N-terminus of the Runt domain, passing below strand βA′ (Figure 4B). All the loops on the lower face of the Runt domain β-barrel, as orientated in Figure 4B (βC-D, βE′-F, the C-terminus and βA′-B), are linked to one another and are well buttressed on one side as a result of the interaction of loop βC-D with CBFβ. Runt domain loop βC-D, which has not been implicated in DNA binding, makes a number of contacts with CBFβ through the side chain of Tyr113. Finally, we find that the three Cys residues in CBFβ (Cys25, Cys107 and Cys124) are not related to the heterodimer interface as proposed (Huang et al., 1999), suggesting that these residues are not directly related to the modulation of oxidation state-dependent behaviour of AML1. Electrostatic surface potential of the Runt domain-CBFβ heterodimer There are two contrasting surfaces on the heterodimer in terms of the electrostatic surface potential (Figure 4Ci and ii). The strongly positive surface corresponds to the position of loops βA′-B, βE′-F and the C-terminus of the Runt domain. This supports the biochemical and human mutation data, which directly implicate these regions of the Runt domain in DNA binding (Lenny et al., 1995; Kagoshima et al., 1996; Osato et al., 1999). In contrast, rotation by 145° from this region reveals a strikingly negative surface (Figure 4Cii), corresponding to the upper outer surface of CBFβ. Five areas on the surface of CBFβ make up this charged surface: the end of strand β6 and the residues prior to helix H5; residues in loop β3-β4; the β5-β6 loop; strand β1; and helix H1. The majority of these residues are conserved in evolution (Figure 3B), suggesting a conserved biological function. The Runt domain-CBFβ interface The Runt domain and CBFβ subunits interact over a large continuous curved interface (Figure 4A), such that a total of 1900 Å2 in solvent-accessible surface area is buried [assuming default radii of GRASP and a 1.4 Å solvent probe (Nicholls et al., 1991)]. The interaction surface of the Runt domain is concave, and packs against a complementary convex surface on CBFβ. The curved heterodimerization surface of the Runt domain shown in Figure 4A involves loops βF-G, βO-A and βB-C at the top of the β-barrel; strands βC, βF and βG; and loop βC-D at the bottom of the β-barrel. The regions of CBFβ involved in heterodimerization are the N-terminal loop and helix H1; strand β1 and loop β1-H3; strands β2, β3 and the connecting loop β2-β3, which together form the central convex interaction surface; strand β4 and the proximal part of loop β4-β5. Figure 5A shows the residues on the surface of CBFβ that are buried in the Runt domain at the interface. Comparing interface residues between human CBFβ and the Drosophila homologues, 13 residues are identical and six involve similar substitutions (Figure 3B). Only two residues have non-conservative substitutions between Drosophila and human. Mutagenesis studies demonstrated a requirement for the N-terminal 5-6 residues of CBFβ for heterodimerization to the Runt domain (Golling et al., 1996; Kagoshima et al., 1996). We find that the Runt domain-CBFβ interaction involves the N-terminus of CBFβ (residues 2-5). The Runt domain loop βO-A, loop βB-C and strand βG are all involved in contacts with the N-terminus of CBFβ, with four potential hydrogen bonding interactions mediated by CBFβ Arg3. Significant chemical shifts at Gln74, Gln79 and Arg83 were noted on binding of a Runt domain-DNA complex to CBFβ (Goger et al., 1999), but the crystal structure demonstrates that this is not a result of burying these residues at the heterodimer interface, and that they do not come to lie proximal to the putative DNA-binding loops βE′-F or βA′-B of the Runt domain (see below). Figure 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). Download figure Download PowerPoint Two-thirds of the residues on the surface of the Runt domain that are buried in the CBFβ subunit (Figure 5B) are either conserved or conservatively substituted between members of the α-subunit family (Figure 3A). Structurally, the conserved cis-Pro156 induces a kink in Runt domain loop βF-G, which makes important contacts with residues in CBFβ. The Runt domain strand βG (residues 159-161) pairs with CBFβ strand β4 (residues 102-104) to form a short antiparallel β-sheet extension. The β-sheet extension between the two subunits is stabilized by a cluster of conserved hydrophobic residues (Runt domain Val159; CBFβ Met101, Ile102 and Leu103). At the opposite end of the heterodimer interface, Runt domain Ser114 and Tyr113 provide a large surface area of interaction with CBFβ. These residues lie in a solvent-accessible polar environment, consistent with previous spectroscopic analysis (Crute et al., 1996). CBFβ residues Glu111 and Asp110 also contribute to this polar environment. Runt domain Met106 makes a significant contribution to the buried surface area in the central part of the heterodimer interface. The functional importance of this residue to the interaction is supported by biochemical studies, which demonstrate that a M106V mutation abolishes the interaction between AML1 and CBFβ in vitro (Akamatsu et al., 1997b). There are a total of 42 contacts between the two subunits, 10 of which represent potential hydrogen-bonding interactions. Bridging contacts are also mediated indirectly via water molecules near the interface. We have identified eight water molecules buried at the heterodimer interface, the role of which is presumably to optimize the complementarity of the interaction interfaces. The structure of the heterodimer interface is consistent with the in vitro Runt domain mutants M106V, G108R and N109D, which show loss of heterodimerization, but preservation of DNA-binding activity (Akamatsu et al., 1997b). Mutations in the Runt domain residues 66-69 located on the βO-A loop also abolish heterodimerization (Lenny et al., 1995). Recent in vivo experiments in Drosophila demonstrated that the G108R Runt protein mutant was dysfunctional in several in vivo assays, showing that the interaction of Runt with the Drosophila CBFβ homologues is essential in vivo for the function of the transcription complex (Li and Gergen, 1999). Homodimer interface of the Runt domain The crystal structure identifies two homodimeric interactions between the Runt domains, one interface between the Runt domains in the dimer of heterodimers, and a smaller interface involving a subset of these interactions within the homodimer QR (890 Å2 solvent-accessible surface area buried as opposed to 1300 Å2). The residues buried at the interface are shown in Figure 5C. The N-terminal residues (59-66) make the most prominent contribution to this interface, which is stabilized further by contacts between strands βA and βB, and loops βB-C and βC′-E. Runt domain Asp66 contributes to both the heterodimer and homodimer interfaces. The homodimeric interaction in the dimer of heterodimers is mediated by a short edge to edge anti-parallel β-sheet formed from the pairing of residues 60-62 at the N-terminal end of the Runt domain, and is stabilized by a hydrophobic cluster involving Pro59, Leu62, Val63, Leu71, Val92 and Leu94 from each subunit. The hydrophobic core of the interface shows strong sequence conservation (Figure 3A). Comparison with NMR data shows conformational differences in the Runt domain Interaction with CBFβ is essential for the in vivo function of AML1 (Wang et al., 1996). In vitro, CBFβ decreases the dissociation constant (Kd) of the isolated Runt domain for its cognate DNA-binding site 5- to 10-fold (Kagoshima et al., 1996). The molecular basis for this enhanced DNA-binding affinity of the Runt domain in the presence of CBFβ is not yet established. The availability of structures of the Runt domain-CBFβ binary complex (this work), together with the co-ordinates for the NMR structures of the Runt domain (performed in the presence of DNA) (Nagata et al., 1999) and the bundle of NMR structures of the uncomplexed CBFβ subunit (Goger et al., 1999; Huang et al., 1999), allows us to address this issue. Although the experimental restraints for the NMR structures are not available, examination of the bundle of NMR structures gives some idea of the flexibility of the structure and the accuracy of the model. We found no significant differences in the crystal structure of CBFβ with respect to the available NMR co-ordinates (Goger et al., 1999; Huang et al., 1999) (PDB codes 2jhb and 1cl3), apart from the disorder in the flexible non-conserved loop β3-β4 (residues 75-80) in the heterodimer. The Cα traces for the structures of the CBFβ- and DNA-bound forms of the Runt domain superimpose well, except in the region of th
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