The leukemia-associated AML1 (Runx1)–CBFβ complex functions

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The mammalian core binding factors (CBFs) are a family ofheterodimeric transcription factors with pivotal roles in normaldevelopment and disease. The proteins in the family contain oneof three different DNA binding α subunits (Runx1–3) and acommon β-subunit, CBFβ. All α-subunits share an evolutionar-ily conserved region of 128 amino acids, known as the Runtdomain, that mediates both DNA binding and heterodimeriza-tion to the β-subunit1. CBFβ stabilizes the DNA-bound state ofthe Runt domain without contacting DNA itself 2. The hetero-dimeric complex between AML1 (also known as Runx1, CBFA2and PEBP2αB) and CBFβ has an essential role in the ontogeny ofdefinitive hematopoiesis3,4 and is the most frequent target forchromosomal rearrangements in human acute leukemias5.

The AML1 gene encodes a 453-amino acid protein, whichincludes the Runt domain between residues 50–177. It was clonedfrom the t(8;21)(q22;q22) in acute myeloid leukemia (AML)6 andwas subsequently shown to be involved in several other recurrentchromosomal translocations, including the t(12;21)(p13;q22)associated with childhood acute lymphoblastic leukemias,t(3;21)(q26;q22) in therapy-related leukemias and myelodyspla-sia and the t(16;21)(q24;q22) found in rare cases of AML7. Thegene encoding CBFβ is also involved in chromosomal transloca-tions in 15% of AML including the inv(16)(p13q22), t(16;16) anddel(16)(q22) (ref. 8). Taken together, the heterodimeric CBFtranscription factor genes AML1 and CBFβ are rearranged in25% of AML and 20% of pediatric common B cell acute lym-phoblastic leukemia7. Acquired somatic mutations in the AML1gene have been reported in sporadic AML and myelodysplasia9–11.Furthermore, AML1 haploinsufficiency protein causes a rarefamilial platelet disorder with propensity to develop AML(FPD/AML)12. The RUNX2 (also known as CBFA1, AML3,PEBP2αA and Osf2) gene is required for bone development13,14,and inherited mutations in one copy of RUNX2 are associatedwith cleidocranial dysplasia (CCD)15.

The consensus binding site for the CBFs has been defined asPyGPyGGTPy (where Py = pyrimidine)2,16, and a high affinitybinding site was described16. The binding site is present in a

nature structural biology • volume 8 number 4 • april 2001 371

number of different viral and cellular promoters and enhancers,including genes specific for hematopoiesis and osteogenesis17.Three-dimensional structures have now been reported for theheterodimerization domain of CBFβ18,19, the Runt domain ofAML120,21 and the heterodimeric complex between the AML1Runt domain and CBFβ22. These studies have shown thatalthough CBFβ has a novel fold, the Runt domain has an s-typeimmunoglobulin (Ig) fold with homology to the DNA bindingdomains of p53, NF-κB, NFAT, the T domain, STAT1 andSTAT3β. The Runt domain has been classified in the SCOP data-base as a member of the p53 family of transcription factors23.CBFβ enhances the DNA binding affinity of the Runt domain∼ 10-fold and binds more efficiently to the DNA-bound Runtdomain than to the Runt domain alone2,24,25.

We have determined the structure of the AML1 Runtdomain–CBFβ–DNA ternary complex in order to explain theconsequences of human disease-associated mutations on DNAbinding and to understand the molecular basis for sequence-spe-cific DNA recognition. Confirmation of the observed specificinteractions was obtained through mutation experiments and bycomparison with previous biochemical data.

The AML1 Runt domain–CBFβ–DNA ternary complexA single AML1 Runt domain–CBFβ heterodimer binds to a 10-base pair (bp) oligonucleotide containing the recognitionsequence by interacting with bases in the major and minorgroove and with the sugar phosphate backbone (Fig. 1). Thedimensions of the complex are ∼ 50 × 62 × 24 Å3. The overall foldof the Runt domain Ig core is consistent with NMR studies per-formed in the presence of DNA20,21 and with X-ray analysis of theDNA-unbound Runt domain–CBFβ complex22. The C-terminal‘tail’ of the Runt domain (residues 170–177) was poorly definedin previous NMR studies because of the lack of NOEs from thissegment to the rest of the protein and the DNA20,21. Our ternarycomplex crystal structure reveals that the extended ‘tail’ andβE′–F loop (‘wing’) elements are ordered in a specific DNA-bound conformation, clamping around the sugar phosphate

The leukemia-associated AML1 (Runx1)–CBFβcomplex functions as a DNA-inducedmolecular clampJerónimo Bravo1, Zhe Li2, Nancy A. Speck2 and Alan J.Warren1

We have determined the structure, at 2.6 Å resolution, of the AML1 (Runx1) Runt domain–CBFβ–DNA ternarycomplex, the most common target for mutations in human leukemia. The structure reveals that the Runt domainDNA binding mechanism is unique within the p53 family of transcription factors. The extended C-terminal ‘tail’and ‘wing’ elements adopt a specific DNA-bound conformation that clamps the phosphate backbone betweenthe major and minor grooves of the distorted B-form DNA recognition site. Furthermore, the extended ‘tail’mediates most of the NF-κB/Rel-like base-specific contacts in the major groove. The structure clearly explains themolecular basis for the loss of DNA binding function of the Runt domain–CBFβ complex as a consequence of thehuman disease-associated mutations in leukemogenesis and cleidocranial dysplasia.

1MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. 2Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire03755, USA.

Correspondence should be addressed to A.J.W. email: [email protected]

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backbone between the major and minor grooves of the DNA(Fig. 1c). The DNA itself is distorted from standard B-formgeometry, bending significantly towards the protein (Table 1).

The 12-stranded β-barrel of the Runt domain is orientatedwith the long axis perpendicular to the DNA helical axis(Fig. 1a–c) with only one face binding the DNA. The βA′–B loop(residues 81–89), the βA′–G′ sheet (βA′ = residues 77–80; βG′ =residues 166–169), the βE′ strand (residues 135–138) and theextended C-terminal ‘tail’ (residues 170–177) dock in the majorgroove and make contacts to the edges of the DNA base pairs andphosphate backbone. The ‘wing’ (residues 139–145) provides anArg that fits into the minor groove. Residues from the βA′–G′sheet, the ‘tail’ and the ‘wing’ form a continuous surface of con-tact, clamping the phosphate backbone between the major andminor grooves of the Runt domain–CBFβ recognition site(Fig. 1c). A total of 1,519 Å2 in solvent-accessible surface area isburied at the protein–DNA interface (assuming default radii ofGRASP and a 1.4 Å solvent probe)26. A significant proportion(44%) of this area is buried by the ‘tail’ contacting the core GGsequence (bases G6, G7) in the 3′ half of the DNA binding site.The structural elements of the Runt domain–CBFβ complexdemonstrated to bind DNA are consistent with NOEs, chemical

372 nature structural biology • volume 8 number 4 • april 2001

shift data and comparisons with other Ig domains20–22,27.However, NOE data suggesting involvement of the βF–G loop21

and Val 74 (ref. 20) are not supported by this structure.CBFβ makes neither direct nor indirect contacts to the DNA

(Fig. 1a–c). Our current model for CBFβ comprises a seven-stranded β-barrel and four peripheral α-helices. An additionalshort β-strand, designated β3′ (residues Tyr 85–Asp 87)(Fig. 1b), extends the antiparallel β-sheet formed by β-strandsβ4, β5 and β6 through hydrogen bonding interactions withstrand β4 (residues Lys 98–Thr 96). Strand β3′ was identifiablefollowing further refinement of the data for the Runtdomain–CBFβ heterodimer. A previous NMR study documentsthe presence of a 310-helix (H4) involving CBFβ residuesArg 83–Arg 90 (ref. 18). The differing secondary structures seenin these studies and the associated high B-factors suggest thatthis region is conformationally flexible.

DNA conformation in the complexThe two DNA duplexes (designated EF and GH) in the asymmet-ric unit of the crystal have nearly identical structures (root meansquare (r.m.s.) deviation of 0.171 Å for 404 atoms). Table 1describes structural parameters per base step of oligonucleotide

Fig. 1 Structure of the AML1 Runt domain–CBFβ–DNA ternarycomplex. Ribbon diagram of the ternary complex, viewed a, perpendicular to the DNA axis and b, parallel to the DNAaxis. The Runt domain is shown in cyan, CBFβ in magenta,DNA strand E in yellow, and DNA strand F in mauve. The ele-ments of the Runt domain that contact the DNA are high-lighted. Strand βA′ and part of loop βA′–B (residues 77–84) arered; loop βE–F (residues 132–145) is green, including the βE′–Floop or ‘wing’ (residues 139–145) and the βE–E′ loop (residues132–138); strand βG′ (residues 166–169) is grey and the ‘tail’(residues 170–177) is blue. Strands and helices are labeled con-sistent with Warren et al.22, except for the additional β-strandin CBFβ, labeled β3′. Numbering corresponds to the aminoacid sequences of human AML16 and CBFβ8. CBFβ residues71–79 are disordered and are shown as a dashed line. Figureswere prepared with the program PREPI. c, Surface representa-tion of the ternary complex. The Runt domain clamps theDNA sugar phosphate backbone between the C-terminal ‘tail’in the major groove and the ‘wing’ in the minor groove. Thefigure was drawn using GRASP26. d, Stereo view of the σA-weighted, solvent flattened electron density map con-toured at 1 σ, showing the interface between Runt domainresidues Arg 80, Asp 171, Arg 174 and Arg 177 and the majorgroove of the DNA. The figure was drawn with Bobscript51.

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EF. The DNA duplex adopts an overall B-DNA-like conforma-tion, with slight underwinding (10.8 base pairs per turn) and anaverage helical rise of 3.27 Å. There are deviations from standardB-DNA geometry, the most striking of which is the marked incli-nation at base pairs G4–C5 (18.8°), G7–T8 (18.5°) and T9–G10(17.3°). These base pair steps have associated high roll values,resulting in significant bending of the DNA towards the protein(by ∼ 40°). The major groove is wide (18.3 Å), and the minorgroove enlarged (13.4 Å) in the region of sequence-specific basecontacts by the Runt domain. This type of DNA structure, notedin other unrelated protein–DNA complexes, has been classifiedas Beg-DNA (where eg means enlarged groove) subclass28. Thus,the DNA conformation in the Runt–CBFβ–DNA complex showssimilarities to the otherwise unrelated glucocorticoid, MetJ,engrailed and Tramtrack protein–DNA complexes28.

Mechanism of DNA recognitionThe protein–DNA interface is well-ordered (Fig. 1d) and the spe-cific interactions are essentially the same for the two indepen-dent ternary complexes in the asymmetric unit of the crystal(r.m.s. deviation of 0.158 Å over 124 atoms in the Runt domain,including the ‘wing’ and ‘tail’) (Figs 1d, 2a,b). The Runt domainis anchored on either side of the major groove through direct andwater-mediated contacts with the regions of the phosphate back-bone that flank and partially overlap the core recognitionsequence on one face of the DNA helix (Fig. 2). At the 5′ end ofthe consensus-binding site, strand βE′ (Arg 135) and loop βA′–B(Lys 83 and Thr 84) contact the phosphate backbone of DNAstrand E (T2, T3 and G4) (Fig. 2a). The phosphate backbone ofDNA strand F (A2, A3, C4 and C5) is contacted by the ‘wing’(Arg 139, Gly 141, Arg 142 and Gly 143), strand βG′ (Ile 168 andThr 169) and the C-terminal ‘tail’ (Val 170) (Fig. 2a). Arg 142(‘wing’) makes additional contacts to bases from both strands inthe minor groove (strand E, G10; strand F, A2). The amino acidsequence between strands βE and βF (Fig. 2b), which includesthe ‘wing’, contains the longest stretch of conserved sequenceidentity in the Runt domain, supporting a critical role for theseresidues in the DNA binding mechanism.


The primary DNA binding specificity determinants are strandβA′ (Arg 80) and the C-terminal ‘tail’ (Asp 171, Arg 174 andArg 177) (Fig. 1d). In particular, the side chain atoms NH1 andNH2 from Arg 80 (strand βA′) form potential bidentate hydro-gen bonding interactions with nitrogen N7 and the exocyclicoxygen O6, respectively, of base G4 (strand E). Two other inter-actions anchor the position of the Arg 80 side chain and guaran-tee specificity. Arg 80 (atom NE1) forms a potential hydrogenbond with the main chain carbonyl oxygen of Val 170 (Fig. 2b).Further, Arg 80 (atom NH1) makes a potential water-mediatedhydrogen bond to base G4 (backbone atom O1P, strand E).Lys 83 makes a similar potential indirect water contact to baseG4 (atom O1P), further stabilizing the position of the Arg 80side chain.

The Runt domain ‘tail’ makes the most extensive set of base con-tacts, which are well-defined in the density (Figs 1d, 2a). Arg 174(atoms NH1 and NH2) makes potential bidentate hydrogen bondsto base G6 of strand E (atoms O6 and N7, respectively). Arg 177(atoms NH1 and NH2) makes potential bidentate hydrogen bondsto base G7 of strand E (atoms N7 and O6, respectively). Asp 171(atoms OD1 and OD2) makes potential hydrogen bonding inter-actions with the complementary bases C4 and C5 (N4 atoms) ofstrand F. Furthermore, Asp 171 makes a potential water-mediatedcontact to base C4 of strand F (backbone atom 01P) and a stabiliz-ing potential salt bridge to Arg 174 (atom NH1). Thus, the orienta-tion of the Arg 174 and Asp 171 side chains is precisely fixed by abuttressing network of hydrogen bonds that further enhances thespecificity of the DNA interaction.

A specific DNA binding conformationOverall, the Cα traces of the DNA-bound and unbound22 crystalforms of the Runt domain–CBFβ complex superimpose well(r.m.s. deviation of 0.71 Å over 242 atoms) (Fig. 3). The regionsof difference correspond to specific elements of the Runt domainin contact with DNA, the ‘tail’ and the ‘wing’. In the absence ofDNA, these elements display high relative B-factor values, sug-gestive of conformational flexibility. In the presence of DNA, the‘tail’ and the ‘wing’ regions are ordered in a specific DNA bind-

Table 1 DNA parameters1

Local base pair step parameters Local base pair helical parameters

Base pair step Slide (Å) Roll (º) Twist (º) X-disp (Å)2 Rise(Å) Incl(º)3 Twist (º)1 GT/AC -0.71 0.08 30.42 -1.36 3.52 0.15 30.662 TT/AA -0.29 4.17 33.09 -1.14 3.05 7.21 33.923 TG/CA 1.27 -2.06 41.61 2.01 3.45 -2.90 41.674 GC/GC -0.14 8.18 24.01 -2.42 2.76 18.83 25.635 CG/CG 0.00 4.68 33.57 -0.83 3.72 7.99 34.346 GG/CC -0.64 7.44 34.61 -2.24 3.46 12.29 35.617 GT/AC -0.86 10.12 30.61 -3.15 2.59 18.55 32.228 TT/AA 0.02 -2.84 35.80 0.47 3.47 -4.62 35.939 TG/CA 0.79 10.74 34.97 0.29 3.43 17.31 36.74

Average values Groove dimensionsmajor minor

Runt-β-DNA -0.06 4.5 33.19 -0.99 3.27 8.31 34.08 18.3 13.4A-DNA -1.53 8.0 31.1 -4.2 2.8 14.6 32.5 12.9 15.8B-DNA 0.23 0.6 36.0 0.1 3.3 2.1 36.5 17.4 10.8

1Parameters were computed with 3DNA (ref. 55) and apply to DNA duplex EF. The parameters for A-form and B-form DNA are based on crystallo-graphic data from the Nucleic Acid Database55.2X-displacement3Inclination

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ing conformation, and the position of the Runt domain ‘tail’ isfixed with respect to both the Ig core and the DNA. There are nosignificant differences in the conformation of the ‘tail’ or ‘wing’regions by comparison of the two independent ternary complex-es in the asymmetric unit of the crystal. These regions are notsubject to packing interactions with neighboring molecules.None of six crystallographically independent copies of the Runtdomain–CBFβ complex in the DNA-unbound form show thearrangement of the ‘tail’ and ‘wing’ seen in the two independentcopies in the ternary complex22. These crystallographic data sug-gest that the DNA provides a template that orders the conforma-tion of the Runt domain ‘tail’ and ‘wing’, supporting previousstudies that indicate structural rearrangement in DNA bindingelements of the Runt domain upon interaction with DNA27,29.

Lack of regular secondary structure within the Runt domain‘wing’ and βC–D loops may be compensated by stabilizing saltbridges and side chain to main chain interactions between theloops. Indeed, polar interactions between the ‘wing’ and theβC–D loop not identifiable in the DNA-unbound form of theRunt domain–CBFβ complex are evident in the DNA-boundform. Thus, Lys 144 (‘wing’) makes potential salt bridges toAsp 110 and Glu 111 (loop βC–D) (Fig. 3). Consistent with animportant structural role for Lys 144, a K144M mutant showsimpaired DNA binding function, while K144R does not30,31.Furthermore, potential hydrogen bonds are identifiable between


the side chain of Asn 109 (βC–D loop) and the carbonyl oxygenof Ser 140 (βE′–F loop). The side chain of Ser 140 contacts thecarbonyl oxygen of Lys 144. Both the main chain and side chain ofSer 140 make potential hydrogen bonds to the main chain of Ile168. These interactions bridge loops βC–D (Asn 109, Asp 110 andGlu 111), βE′–F (Ser 140 and Lys 144) and the parallel βA′–G′sheet (Ile 168). The functional importance of these interactions issupported by mutants S140G and N109D, which show loss ofDNA binding and heterodimerization32. Moreover, the mutationS140N is described in a patient with CCD33.

Fig. 2 DNA recognition mechanism. a, Polar (red) and hydrophobic (blue) interactions between the DNA and the protein. DNA strand E is shown inyellow and strand F in mauve. The central six base pairs corresponding to the consensus DNA binding sequence are highlighted in bold. b, Ribbondiagram of the interactions between the Runt domain and DNA. Structural elements involved in DNA binding are highlighted as in Fig. 1. Residuesmutated in human disease that contact DNA are enclosed in black boxes. Polar interactions are indicated by dashed lines. The included water mole-cules (red spheres) are present in both copies of the complex. Figure was prepared with MOLSCRIPT52.

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Fig. 3 DNA-induced ordering of the ‘tail’ and ‘wing’. Least squares super-position of the Cα traces of the binary Runt domain–CBFβ crystal struc-ture (PDB accession code 1E50) onto the ternary Runt domain–CBFβ–DNAcomplex crystal structure. In the ternary complex, the Runt domain iscyan, and CBFβ is magenta; in the binary complex, the Runt domain isbrown, and CBFβ is yellow. The two regions that undergo DNA-inducedordering (‘wing’ and ‘tail’) are marked by arrows. Gly 143 (‘wing’) showsthe largest shift (7 Å) in the relative position of any Cα.

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These data suggest that stabilization of Runt domain loopsβC–D and βE′–F is an important determinant of the affinity of theinteraction between the Runt domain and DNA. Stabilization ofthe specific DNA-bound conformation of these loops may be pro-vided by the interaction with CBFβ. p53 uses a bound zinc atom,in addition to salt bridge interactions, to stabilize the loops that arestructurally analogous to the Runt domain βC–D loop and‘wing’34. Comparison of the DNA-bound and unbound Runtdomain–CBFβ complexes identifies local conformational changesbetween CBFβ loop β2–3 and Runt domain loop βC–D (Fig. 3). Inparticular, the change in conformation of the bulky aromatic sidechain of Tyr 113 (which is buried at the interface between the Runtdomain and CBFβ) is accommodated by reorientation of the sidechain of CBFβ Asn 63. Because CBFβ loop β2–3 makes an impor-tant energetic and functional contribution to the heterodimerinterface24, these conformational changes offer a possible explana-tion for the change in the free energy of interaction between CBFβand the Runt domain in the presence and absence of DNA.

Mutational analysisWe confirmed the functional relevance of the specific interactionsobserved in our structure by substituting the implicated aminoacids with Ala. The mutant Runt domain proteins tested includeresidues 41–214 of the murine AML1 protein (which extends 9residues N-terminal and 37 residues C-terminal to the conservedRunt domain sequence). Mutant Runt domains were assayed fortheir ability to interact with a high affinity consensus DNA bind-ing site in a yeast one-hybrid assay by expressing them as fusionsto the GAL4 activation domain. The plasmids expressing thefusion proteins were transformed into a yeast strain containing alacZ reporter gene under the regulation of three tandem repeatsof a high affinity binding site; both filter and liquid β-galactosidase assays were performed (Fig. 4a,b). Ala substitu-tion of all side chains directly contacting DNA bases or the phos-phate backbone in our structure (Arg 80, Arg 135, Arg 139,Arg 142, Lys 167, Asp 171, Arg 174 and Arg 177) severely perturbDNA binding, reducing it to background levels in the yeast assays.


Two side chains participating in water-mediated contacts, Lys 83and Thr 169, were also sensitive to Ala substitution. Substitutionof Thr 84 with Ala did not affect DNA binding. However, thewater-mediated contact by Thr 84 to DNA was only observed inone of the two ternary complexes in the crystal. The loss of DNAbinding activity by Runt domain mutants R80A, K83A, R139A,R174A and R177A in the yeast one-hydrid assay is consistent withpublished biochemical data for the mutants R80C9, K83N9,R139Q35, R139G11, R174Q36 and R177Q9.

To confirm the importance of the other five side chains thatcontact DNA in the structure (Arg 135, Arg 142, Lys 167, Thr 169and Asp 171) we purified proteins containing Ala substitutionsfor these residues and tested their activity in electrophoreticmobility shift assays (EMSA). Ala substitution of all five residues,particularly R142A (‘wing’), perturbed DNA binding. DecreasedDNA binding by the R135A, R142A, T169A and D171A mutantswas observed at Runt domain concentrations 100-fold higherthan the dissociation constant (Kd) for the wild type Runtdomain–DNA complex (Fig. 4c). The perturbation caused by theK167A mutation was only apparent at protein concentrations ator below the Kd of the wild type Runt domain–DNA complex(not shown), indicating that the Kd increased by <100-fold.CBFβ, when added at a concentration two-fold > Kd for the Runtdomain–DNA complex, shifted ∼ 50% of the wild type Runtdomain–DNA complex into a ternary Runt domain–CBFβ–DNAcomplex. A Runt domain–CBFβ–DNA complex was alsoobserved for all of the Runt domain mutants (the complexformed with R142A is visible at darker exposures, not shown),indicating that none of the mutations substantially perturbed theassociation of CBFβ.

Consistency with biochemical dataThe DNA contacts demonstrated in the crystal structure of theAML1 Runt domain–CBFβ–DNA ternary complex (Fig. 2a)account for most of the observed effects of chemical modifica-tions on the DNA2,37–39. Bases pairs including T3, C5 and T8(strand E) are not in direct contact with protein, which agrees

Fig. 4 In vitro DNA binding assays. a, Filter β-galactosidase assays. The GAL4-Runt domain fusionderivatives carried in the yeast transformants are indicated. Six independent colonies were streakedin each segment. b, Quantitative liquid β-galactosidase assays. c, Binding assays. Runt domain con-centrations (2.2. × 10–9 M) 100-fold > Kd for the wild type Runt domain–DNA complex25 were used forthe wild type (WT) Runt domain (RD) and the Ala-substituted proteins. The DNA concentration is 2.2 × 10–8 M. CBFβ was added to alternate lanes at a concentration 2-fold > Kd for the RD–DNA com-plex24,25. Squares mark the position of single stranded (SS) DNA, free double stranded (DS) DNA andthe protein–DNA complexes. Faster migrating protein–DNA bands represent complexes formed fromminor Runt domain degradation products.

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with the sequence variability of the DNA in natural target sites atthese positions. The selection for pyrimidine residues at thesepositions may reflect ‘indirect’ preferences for bases that allowbending of the DNA, supported by the high degree of roll and theinclination observed at these base steps (Table 1). The observeddistortion and bending of the DNA is consistent with circularpermutation40 and circular dichroism spectroscopy25 analyses.The Kd for DNA binding (in the range of 10–10–10–12 M) is low,despite that the Runt domain itself only binds as a monomer.This may be explained by the large accessible surface area (1,519 Å2) buried upon complex formation and the large num-ber of direct base contacts (a total of 10). All the critical sidechain to DNA contacts are made by evolutionarily invariantresidues in comparative sequence alignment of Runt domainproteins22. This suggests that the DNA binding mechanismrevealed by the AML1 Runt domain–CBFβ–DNA structure isgeneral to the entire family of Runt domain proteins.


Many disease-related mutations affect DNA contactsA key finding that emerges from the structure of the ternarycomplex is that most of the disease-associated missense muta-tions that map to the Runt domain affect residues involved insequence-specific base interactions or DNA backbone contacts(Fig. 2b). Leukemia-associated mutations target most of theresidues that contact DNA in the structure: R80C, K83N, R135G,R139Q, D171G, R174Q, R174G, R177G, R177Q and R177ter(ter denotes a mutation of Arg 177 to a termination codon). Allof these mutations would potentially disrupt critical hydrogenbonding interactions to the DNA backbone (K83N, R135G,R139Q) or bases (R80C, D171G, R174Q, R174G, R177G,R177Q, R177ter). The mutation R80C was identified in a patientwith blast transformation of chronic myeloid leukemia andK83N in a patient with relapsed AML M3 (ref. 9). The R174Qmutation was reported in the context of FPD/AML12, andArg 174 was the residue in CBFA1 (Runx2/AML3) that was mostfrequently affected by congenital mutations (including R174Q,R174W) in CCD35,36. R135G, G138D and R171G were describedin MoAML10, whereas R139Q was reported in both FPD/AMLand CCD12,35,36. The G138D mutation would be expected to dis-rupt the conformation of the βE′–F and βC–D loops, thus dis-rupting DNA binding. Several other mutations are described inthe context of CCD: M124R, S140N, F146S, K167N33,41,42 andT169I (H. Kanegane, pers. comm.). Impaired DNA binding byK167N and T169I is explained by disruption of DNA backbonecontacts (Fig. 2). The mutations L148F, F146S and M124R wouldbe expected to destabilize the overall fold of the Runt domainbecause they affect residues projecting into the hydrophobic coreof the structure; Ser 140 has an important structural role, as dis-cussed above. In summary, the structure of the AML1 Runtdomain–CBFβ–DNA ternary complex clearly explains the mole-cular consequences of the human disease-associated mutationson DNA binding.

Rel-like DNA contacts by the ‘tail’Structurally, the AML1 Runt domain belongs to the p53 familyof transcription factors, with closest homology to STAT3β22. Incommon with the other members of the family, the Runt domaincontacts DNA using loops from a single face of the β-barrel core(Fig. 1). However, the ternary Runt domain–CBFβ–DNA com-plex demonstrates that the Runt domain employs a distinctivevariation in the DNA recognition mechanism because most ofthe base contacts in the major groove are made from the unusualextended C-terminal ‘tail’ projecting from the core Ig β-barrel.By contrast, the p53 DNA binding domain34 and the Brachyury Tdomain43 have C-terminal α-helical extensions from the Ig corewhich are used to interact with the major (p53) or the minor(T domain) grooves of the DNA. The mapping of human dis-ease-associated mutations to this region together with mutagen-esis data described in this work and in previous reports9,36

demonstrate the functional and biological importance ofresidues within the Runt domain ‘tail’ for DNA recognition.

Despite the lack of sequence homology and the use of differ-ent structural elements for sequence-specific DNA binding, the

Fig. 5 Rel/NFκB-like DNA contacts by the Runt domain. Specific base con-tacts are indicated by arrows for the human AML1 Runt domain (thiswork), mouse p65 (ref. 53), Gambif1 (ref. 44) and NFAT54. The buttressinghydrogen bonds made by the conserved Glu or Asp (Runt domain) areindicated (dashed lines). The conserved GG core elements are highlight-ed in yellow.

Table 2 Data collection and refinement statistics

Data collection statisticsResolution 2.6 ÅObservation / unique reflections 193,723 / 27,926Completeness (%)1 99.1 (98.8)Rsym

1,2 0.126 (0.380)< I/σ >1 19.46 (2.2)

Structure refinement statisticsResolution 50–2.6 ÅNumber of atoms

Protein 4,005Nucleic acid 808Waters 56

Rcryst3 0.23

Rfree3 0.27 (8.0)

R.m.s. deviation from ideality4

Bonds (Å) 0.017 Angles (°) 1.9Dihedrals (°) 24.3

1Values in parentheses are for the highest resolution shell.2Rsym = ΣhklΣi | Ii (hkl) – <I (hkl)> | / ΣhklΣi Ii (hkl)3Rcryst and Rfree = Σ |Fobs – Fcalc| / Σ Fobs; Rfree calculated with the percentageof the data shown in parentheses.4R.m.s. deviations for bond angles and lengths in regard to Engh & Huberparameters.

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crystal structure of the ternary complex reveals remarkable con-servation in the specific base contacts used with the class II Relfamily of proteins (Fig. 5). This family is typified by NF-κB, p65,RelB, c-Rel, the malarial protein Gambif1 and the DrosophilaDorsal and Dif proteins, which, like AML1, contain transcrip-tional activation domains and have important roles in develop-ment44. Three guanine bases (G4, G6 and G7) are conserved inall CBF recognition sites identified2,37,38. The Rel proteins con-tact a core GG sequence within their recognition sites usingthree conserved residues (two Arg and a Glu) from the ABrecognition loop of a Rel monomer (Fig. 5). The Runt domainalso contacts a core GG sequence using two conserved Arg andan Asp residue from the ‘tail’ (Figs 2b, 5), and uses a single Argfrom the βA′ strand to contact the third guanine base 5′ to thecore GG sequence. At present, the evolutionary relationshipbetween the different members of the p53 family of transcrip-tion factors is unclear45.

MethodsProtein and DNA purification. A fragment of human AML1(residues 50–183 out of 453), including the conserved Runt domain(residues 50–177) and the heterodimerization domain of CBFβ(residues 1–135), was expressed and purified from Escherichia coli 22.Two 10-bp oligonucleotides, 5′-GTTGCGGTTG-3′ and 5′-CAACCG-CAAC-3′ (lacking the 5′ phosphates) were synthesized commercially(MWG-Biotech) and annealed in 10 mM Tris, pH 7.4, 1 mM EDTA,100 mM NaCl. The AML1 Runt domain–CBFβ–DNA ternary complexwas prepared in a buffer containing 200 mM NaCl, 20 mM 2-(N-mor-pholino)ethanesulphonic acid (MES), pH 6.0, 5 mM dithiothreitol(DTT), 1 mM EDTA by mixing protein with a 10% molar excess ofDNA (around 70 µM). The protein–DNA complex was purified on aHiPrep Sephacryl S100 (Pharmacia) column in a buffer containing300 mM NaCl, 20 mM MES, pH 6.0, 5 mM DTT and 1 mM EDTA; con-centrated to 0.13 mM and stored in liquid nitrogen.

Crystallization and data collection. AML1 Runtdomain–CBFβ–DNA cocrystals were grown at 23 °C using the hang-ing drop vapor diffusion method. Drops containing 1 µlprotein–DNA complex and 1 µl of crystallization buffer (50 mMHepes, pH 6.5, 3% (w/v) PEG 8000 (Hampton), 5 mM DTT) were equi-librated against 1 ml of reservoir solution containing the same crys-tallization buffer. Crystals grew over a week to ∼ 400 × 70 × 70 µm3.Prior to data collection, crystals were harvested by stepwise transferto a cryobuffer (35% (v/v) glycerol, 200 mM NaCl, 100 mM Hepes,pH 6.5, 7% (w/v) PEG 8000) and cryocooled in liquid nitrogen. Thecrystals belong to the P43212 space group, with unit cell parametersa = 115.0 Å, c = 133.9 Å. They have a solvent content of 56% withtwo ternary complexes in the asymmetric unit. Diffraction datawere collected with cryocooling at 100 K at ESRF beamline ID14-3,Grenoble, using a MAR CCD detector (Table 2). Data were integrat-ed with MOSFLM46 and scaled with Scala47.


Structure determination. An initial rotation/translation searchwas carried out with the program AmoRe47 using our solved struc-ture of the AML1 Runt domain–CBFβ complex22 (PDB accession code1E50). A second search with a DNA model of the c-fos promoter (PDBaccession code 1BC7) located the DNA molecules. During the refine-ment, restrained noncrystallographic symmetry was maintained withweights of 300 for the protein and 100 for the DNA. The model wasrefined with the program CNS48 followed by manual rebuilding inO49 using solvent flattened maps. The structure has been refined to aRfree factor of 0.27 with no residues in the disallowed regions of theRamachandran plot. Residues 50–53 and 178–183 (C-terminal to theconserved Runt domain) of the AML1 fragment are disordered. Nodensity was evident for residues 71–79 of CBFβ.

Alanine substitutions. DNA sequence corresponding to aminoacid residues 41–214 of the murine AML1 protein (extending nineresidues N-terminal and 36 residues beyond the C-terminus of theAML1 fragment used in the structure determination) was subclonedinto the pBluescript SK+ (Stratagene) vector. Ala substitutions weremade using the Stratagene QuikChange mutagenesis kit followingthe manufacturer’s protocols.

Yeast one-hybrid assays. Yeast one-hybrid assays were carriedout using the Clontech MATCHMAKER Yeast One-Hybrid System.Wild type and Ala-substituted Runt domains (amino acids 41–214)were subcloned into the yeast expression vector pGAD424 (GAL4activation domain/LEU2 marker) and transformed into the S. cere-visiae YM4271(HA) strain by lithium acetate-TE50. The YM4271(HA)strain is derived from YM4271 but contains a lacZ reporter geneunder the regulation of three tandem repeats of a high-affinitycore site (5′-GAATTC-(TTTGCGGTTAG)3-GTCGAC-3′) (ref. 16). β-galactosidase assays were performed following the manufactur-er’s protocols (Clontech).

Binding assays. The Runt domain (amino acids 41–214) and CBFβ(amino acids 1–141) proteins were purified19,21 and EMSAs per-formed as described25, using an 18 bp DNA sequence containing thecore site from the SL3-3 murine leukemia virus enhancer.

Coordinates. The coordinates have been deposited in the ProteinData Bank (accession code 1H9D).

AcknowledgmentsWe thank S. Arzt for help with data collection at ESRF beamline ID14-3; R.Williams for invaluable assistance; S.A. Islam and M.J.E. Sternberg (ICRF) for theprogram PREPI; J. Bushweller and H. Kanegane for sharing unpublished data; C.Müller for the preprint of his review; D. Rhodes, A. Murzin, D. Neuhaus and A.Travers for helpful discussions. A.J.W. is supported by an MRC Senior ClinicalFellowship. N.A.S. is supported by Public Health Service grants.

Received 26 January, 2001; accepted 5 March, 2001.

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The leukemia-associated AML1 (Runx1)–CBFβ complex functions

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