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Originally published In Press as doi:10.1074/jbc.M303973200 on July 9, 2003 Originally published In Press as doi:10.1074/jbc.M303973200 on June 13, 2003

J. Biol. Chem., Vol. 278, Issue 35, 33088-33096, August 29, 2003
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Energetic Contribution of Residues in the Runx1 Runt Domain to DNA Binding*

Zhe Li {ddagger}, Jiangli Yan §, Christina J. Matheny {ddagger} , Takeshi Corpora §, Jerónimo Bravo || **, Alan J. Warren ||, John H. Bushweller § and Nancy A. Speck {ddagger} {ddagger}{ddagger}

From the {ddagger}Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, the §Department of Molecular Physiology and Biological Physics and Department of Chemistry, University of Virginia, Charlottesville, Virginia 22906-0011, and ||MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

Received for publication, April 15, 2003 , and in revised form, June 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in hematopoiesis and in the development of bone, stomach epithelium, and proprioceptive neurons. Mutations in CBF genes are found in leukemias, bone disorders, and gastric cancer. CBFs consist of a DNA-binding CBF{alpha} subunit and a non-DNA-binding CBF{beta} subunit. DNA binding and heterodimerization with CBF{beta} are mediated by the Runt domain in CBF{alpha}. Here we report an alanine-scanning mutagenesis study of the Runt domain that targeted amino acids identified by structural studies to reside at the DNA or CBF{beta} interface, as well as amino acids mutated in human disease. We determined the energy contributed by each of the DNA-contacting residues in the Runt domain to DNA binding both in the absence and presence of CBF{beta}. We propose mechanisms by which mutations in the Runt domain found in hematopoietic and bone disorders affect its affinity for DNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The core-binding factors (CBFs)1 are a small family of heterodimeric transcription factors that play critical roles both in mammalian development and in human disease. CBF{alpha} subunits are encoded by three related genes, RUNX1, RUNX2, and RUNX3 (1–3), whereas the common CBF{beta} subunit is encoded by a single gene called CBFB (4, 5). Genetic loss- and gain-of-function studies in mice documented roles for the CBF proteins in at least four developmental processes. Runx1 and Cbfb are required for definitive hematopoiesis (6–9), and both Runx2 and Cbfb are required for bone formation (10–14). The Runx3 gene is required for proper development of the stomach epithelium (15), for the development of a subset of sensory neurons (16, 17), and for epigenetic silencing of the CD4 gene in T lymphocytes (18). Lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer (15).

RUNX1 and CBFB are frequent targets of mutations in human leukemias (19, 20). RUNX1 is disrupted by the t(8; 21)(q22;q22) in 15% of de novo acute myeloid leukemia (AML M2 subtype) (2, 21), the t(12;21)(p13;q22) in 30% of pediatric de novo acute lymphoblastic leukemia (22–25), and by the relatively rare t(1;21)(p36;q22), t(3;21)(q26;q22), t(5;21)(q13;q22), t(12;21)(q24;q22), t(14;21)(q22;q22), t(15;21)(q22;q22), t(16; 21)(q24;q22), and t(17;21)(q11.2;q22) in the therapy-related leukemias and myelodysplasias (MDS) (2, 22, 23, 26–29). Biallelic point mutations in RUNX1 are found in AML of the M0 subtype (30–32). RUNX1 mutations in AML M0 include both nonsense mutations that introduce premature termination codons and missense mutations. Monoallelic point mutations in RUNX1 were identified in AML M0, MDS, pediatric acute lymphoblastic leukemia, and chronic myelogenous leukemia in blast crisis (30–33). In addition, haploinsufficiency of the RUNX1 gene is responsible for a rare familial platelet disorder with propensity for acute myelogenous leukemia (FPD/AML) (34–36).

The RUNX2 gene is also the target of mutations that are responsible for an inherited human skeletal disorder called cleidocranial dysplasia (CCD), which is characterized by moderate skeletal malformations including delayed closure of the fontanelles, hypoplastic or absent clavicles, short stature, and supernumerary teeth (37). Mutations in CCD have been identified throughout the RUNX2 gene (37–40); however, many of the point mutations found in RUNX2 and most of the mutations in RUNX1 found in hematopoietic disease are clustered in the DNA-binding "Runt" domain.

The Runt domain is a 118-amino acid domain that is responsible for both DNA binding as well as for heterodimerizing with CBF{beta} (41). The three-dimensional structures of the isolated Runt domain, a Runt domain-DNA complex, a Runt domain-CBF{beta} complex, and the Runt domain-CBF{beta}-DNA complex have been determined (42–48). The Runt domain is an s-type immunoglobulin-fold similar to that of the DNA-binding domains of NFAT, STAT, p53, and NF{kappa}B (49). The crystal structures of Runt domain-DNA complexes reveal that the Runt domain uses two loops ({beta}A'-B, {beta}E'-F), one {beta}-sheet ({beta}A'-G'), one {beta}-strand ({beta}E'), and the C-terminal tail to contact DNA (45, 47). The extended C-terminal "tail" and the {beta}E'-F loop ("wing") elements adopt a specific conformation in the ternary complex that clamps the phosphate backbone between the major and minor grooves of the DNA recognition site (45, 47).

Previous biochemical data and mutagenesis studies of the Runt domain have confirmed many of the specific interactions between DNA and the Runt domain observed in the crystal structures (30, 35, 40, 45, 47, 50, 51). However, no studies have yet systematically determined the energetic contribution of each of the amino acid side chains to DNA binding. Here we report an extensive alanine-scanning mutagenesis study of the Runt domain, targeting those amino acids near the DNA binding interface as well as amino acids mutated in disease. We also measured the energetic contributions of 10 DNA-contacting residues in the Runt domain to DNA binding, in the absence and presence of CBF{beta}. We confirmed that all 10 residues contribute to DNA binding energetically. Among them, we found that amino acid Arg-174, the residue most frequently mutated in AML M0, FPD/AML, and CCD, contributes the greatest binding energy to the interaction with DNA. We confirmed that the Runt domain is very sensitive to amino acid substitutions, and our mutagenesis study provides a rationale for how mutations in the Runt domain affect DNA binding. Overall, the data reported here lend support to and further refine the Runt domain structure determined by NMR spectroscopy and x-ray crystallography.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Site-directed Mutagenesis—We PCR-amplified a fragment encoding the open reading frame of murine Runx1 (amino acids 41–214) using the forward primer 5'-ATC GAA TTC CAT ATG GCC AGC AAG CTG AGG AGC GGC GAC-3' and the reverse primer 5'-AGC CGG ATC CTA GTG CGG GCT GAC CCT-3'. We added EcoRI and NdeI restriction sites (underlined) to the 5' end of the fragment by the forward primer, and a BamHI site (underlined) to the 3' end by the reverse primer. We then subcloned the PCR-amplified fragment into the pBluescript SK+ vector (Stratagene) between its EcoRI and BamHI sites. Site-directed alanine replacement mutagenesis (if the amino acid was alanine it was changed to glycine) was performed on this plasmid using the Stratagene QuickChange mutagenesis kit following the manufacturer's protocols.

Yeast One-hybrid and Two-hybrid Assays—We subcloned the mutated Runt domain fragments into the yeast expression vector pGAD424 (Clontech, GAL4 activation domain/LEU2 marker) between the EcoRI and BamHI sites. We transformed the pGAD424 vector containing either the wild type or alanine-substituted Runt domain fragments into the Saccharomyces cerevisiae strain YM4271(HA) or YM4271(MLV) by the lithium acetate/TE protocol of Schiestl and Gietz (52). Both YM4271(HA) and YM4271(MLV) are derived from YM4271 and contain a lacZ reporter gene under the regulation of three tandem repeats of either a high affinity (HA) Core site (5'-GAATTC(TTTGCGGTTAG)3GTCGAC-3') or a lower affinity Moloney murine leukemia virus (MLV) Core site (5'-GAATTC(TCTGTGGTAAG)3GTCGAC-3') (53, 54). We performed {beta}-galactosidase assays following the protocols provided in the MATCHMAKER Yeast One-hybrid system (Clontech). We performed the yeast two-hybrid assay using the same pGAD424 vector containing either the wild type or alanine-substituted Runt domain fragments together with a plasmid based on pGBT9 (GAL4 DNA-binding domain/TRP marker) that contains a cDNA encoding the full-length murine CBF{beta} protein (55) co-transformed into the S. cerevisiae strain Y153.

Purification of Runt Domain Mutants—We subcloned NdeI-BamHI fragments encoding wild type and mutated Runt domains from either the pBluescript SK+ vector or the pGAD424 vector into the bacterial pET-3c (Novagen) expression vector, and we purified individual 15N-labeled mutant proteins as described previously (44, 56).

NMR Spectroscopy—All measurements were performed on a Varian Inova 500 MHz NMR spectrometer equipped with an actively shielded triple resonance probe from Nalorac Corp. We prepared samples of Runt domain and Runt domain-DNA complexes and recorded 15N-1H HSQC spectra at 40 °C as described previously (44).

Urea Denaturation Monitored by Fluorescence Spectroscopy—We detected the urea denaturation of the Runt domain by tryptophan fluorescence as described previously (56). All fluorescence experiments were performed using a protein concentration of 20–40 µM in a total volume of 0.8 ml in 25 mM Tris-HCl (pH 7.5), 50 µM EDTA, and 1 mM dithiothreitol. Fluorescence measurements were performed on a SPEX Fluoromax spectrofluorometer (Spex Industries, Edison, NJ). Spectra were recorded in the range of 300–370 nm with an increment of 4 nm and an integration time of 1 s. Two scans were averaged to minimize error, and the background spectrum of the buffer alone was taken first for later subtraction. The samples were excited at a wavelength of 280 nm, and the emission was detected at 340 nm. To follow the denaturation of the Runt domain, individual 0.8-ml solutions of the Runt domain (20–40 µM) were incubated with increasing concentrations of urea (0–6.0 M), and the samples were equilibrated for 2 h at 4 °C prior to fluorescence measurement.

The parameters for the unfolding curves were obtained by nonlinear, least squares fitting using two-state equations (Equation 1 and Equation 2) as described previously (57–59).

(Eq. 1)

(Eq. 2)
The data were fit using Equation 1, where Fobs is the fluorescence observed; {alpha}N and {alpha}D are the intercepts; {beta}N and {beta}D are the slopes of the base lines at low (N) and high (D) denaturant concentrations, respectively; R is the gas constant; T is the absolute temperature in K; [D] is the denaturant concentration; [D]50% is the concentration of the denaturant when half of the protein is denatured; and m is the slope of the unfolding transition. We used Equation 2 to determine {Delta}GN-D, where Fobs is the fluorescence observed; {alpha}N and {alpha}D are the intercepts; {beta}N and {beta}D are the slopes of the base lines at low (N) and high (D) denaturant concentrations, respectively; R is the gas constant; T is the absolute temperature in K; [D] is the denaturant concentration; {Delta}GN-D is the free energy of unfolding in the absence of denaturant; and m is the slope of the transition. We used Origin 5.0 software for non-linear least square fitting.

Equilibrium Binding Constant Measurements—We used electrophoretic mobility shift assays (EMSA) to measure equilibrium binding constants (K2 and K4) of the wild type and alanine-substituted Runt domain proteins and an 18-bp DNA sequence containing the Core site from the SL3-3 murine leukemia virus enhancer (5'-GGATATCTGTGGTTAAGCA-3') (55, 60). We define K2 as the Kd of the Runt domain for DNA, and K4 is the affinity of the Runt domain-CBF{beta} heterodimer for DNA (55, 60) (Fig. 4). {Delta}G0, {Delta}{Delta}G0, and their standard errors were calculated as described previously (55).



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  		FIG. 4.
Equilibrium binding constant measurements. A, schematic diagram of the potential interactions between the Runt domain (RD), CBF{beta} ({beta}), and DNA. B, EMSAs with the wild type Runt domain (left panels) and RD(R80A) (right panels) titrated onto DNA, in the absence or presence of CBF{beta}. Positions of single-stranded (ss) DNA, double-stranded (ds) DNA, and the binary RD-DNA and ternary RD-CBF{beta}-DNA complexes are indicated.

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Alanine-scanning Mutagenesis of the Runt Domain—We performed an alanine-scanning mutagenesis of the Runt domain to identify and characterize its DNA-binding and heterodimerization interfaces, and to assess the sensitivity of the Runt domain fold to amino acid substitutions. We chose amino acids to mutate based on five criteria. We first targeted 27 amino acids in the Runt domain that displayed nuclear Overhauser effects between their backbone NH groups and the DNA, because this was the only structural information available to us at the time we began the study (44). We also mutated amino acids identified by a chemical shift perturbation analysis of the Runt domain performed in the absence and presence of DNA that similarly examined backbone and side chain NH protons (61). We mutated DNA-contacting amino acids implicated by the crystal structures of the Runt domain-DNA and the Runt domain-CBF{beta}-DNA complexes once those structures were available (45, 47) (Fig. 2A). Some of the residues implicated by the crystal structures had been identified in the NMR studies, but five (Arg-135, Arg-142, Gly-143, Lys-167, and Arg-174) had not. We made alanine substitutions in all residues thought to reside at the CBF{beta} interface, as identified by a chemical shift perturbation study (44) and in the crystal structures (45, 47, 48). Finally, we replaced all residues that are mutated in the human RUNX1 and RUNX2 proteins in AML M0, FPD/AML, MDS, and CCD patients. In total we substituted 74 of the 118 amino acids in the Runt domain (amino acids 61–178) with alanines (Table I,Table I).



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  		FIG. 2.
Results of yeast one-hybrid and two-hybrid assays. A, structure of the Runt domain showing the location of amino acids that contact DNA (side chains in red) and CBF{beta} (side chains in green) according to the crystal structures (45, 47, 48). Also shown in gray are the amino acids that are stabilized in different conformations in the absence and presence of CBF{beta} and DNA. B, graphic view of the results of the yeast one-hybrid assay detecting DNA binding activity. Gold (and labeled), mutations that severely disrupted DNA binding (++ or – in Table I,Table I); pink, mutations that moderately affected DNA binding (+/– or +); gray, mutations that did not affect DNA binding. C, results of the yeast two-hybrid assay that detects heterodimerization with CBF{beta}. Gold, mutations that severely affected CBF{beta} binding; pink, mutations that moderately affected CBF{beta} binding; gray, mutations that did not affect CBF{beta} binding. D, classification of mutants based on the proposed mechanisms by which DNA binding is disrupted. Gold, mutations that disrupt direct DNA contacts; gray (and labeled), mutations that disrupt salt bridges or hydrogen bonds mediated by the side chain; pink, mutations that either locally or globally perturb the Runt domain fold.

 

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  		TABLE I
Summary of limited alanine scanning mutagenesis of the Runt domain

 

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  		TABLE I (continued)
Summary of limited alanine scanning mutagenesis of the Runt domain

 

We characterized the effects of these alanine substitutions on DNA binding using a yeast one-hybrid assay (Fig. 1A). We generated a yeast reporter strain containing three high affinity (HA) CBF binding (Core) sites driving the bacterial lacZ gene. We also developed a reporter strain that contains three lower affinity CBF binding sites from the Moloney MLV enhancer driving lacZ (Fig. 1A). We fused the Runt domain (RD) in-frame to the GAL4 activation domain (GAL4AD) and analyzed binding of the fusion protein to the Core sites in both reporter strains by visualizing {beta}-galactosidase activity. Expression of the wild type GAL4AD-RD fusion protein results in a robust {beta}-galactosidase signal in the reporter strain containing HA Core sites and a barely detectable signal in the reporter strain with the lower affinity MLV Core sites (Fig. 1A). The difference in Runt domain binding affinity for the HA and MLV Core sites is ~10-fold (2.7 x 10–12 versus 2.9 x 10–11 M) (54); thus our yeast one-hybrid assay is sensitive in a 10-fold range of DNA binding affinity. For all of the yeast one-hybrid assays presented in Table I,Table I, we used the reporter strain containing the HA Core sites.



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  		FIG. 1.
Analyses of Runt domain alanine mutants using yeast one-hybrid and two-hybrid assays. A, schematic diagram of the yeast one-hybrid assay. The yeast strains have a lacZ reporter gene under the control of three repeats of either high affinity (HA) Core sites or Moloney murine leukemia virus (MLV) Core sites. {beta}-Galactosidase filter assays are shown below. B, schematic diagram of the yeast two-hybrid assay. The Runt domains were fused to the GAL4AD. A full-length murine CBF{beta} was fused to the GAL4DBD. The reporter strain has the lacZ gene under the control of the yeast GAL upstream activating sequence.

 

We also analyzed the effects of the alanine substitutions on heterodimerization with CBF{beta} using a yeast two-hybrid assay (Fig. 1B). This assay utilized the same GAL4AD-RD fusion protein employed in the yeast one-hybrid assay, in conjunction with a GAL4 DNA-binding domain CBF{beta} fusion protein (GAL4DBD-CBF{beta}) to drive transcription from GAL4-binding sites. The crystal structures indicate that non-overlapping sets of amino acids are involved in DNA and CBF{beta} binding (45, 47). Therefore, any alanine substitution that affects activity in either the yeast one- or two-hybrid assay that does not involve a residue known to directly contact either DNA or CBF{beta} has in all likelihood locally or globally affected the Runt domain fold. We cannot, however, rule out the possibility that a reduction in expression levels in yeast caused by factors unrelated to protein stability or folding might contribute to a decreased activity in both yeast one- and two-hybrid assays.

Alanine substitution of 21 of the Runt domain residues severely affected DNA binding according to the yeast one-hybrid assay (Table I,Table I, "++" and "–" scores). These residues are displayed in Fig. 2B on the Runt domain fold. Ten of these amino acids (Arg-80, Lys-83, Arg-135, Arg-139, Arg-142, Lys-167, Thr-169, Asp-171, Arg-174, and Arg-177) were shown in the crystal structures of the RD-CBF{beta}-DNA ternary complex to make side chain contacts to the DNA bases or the phosphate backbone (45, 47). Alanine substitution of all of these amino acids specifically affected DNA binding but not heterodimerization with CBF{beta}, as measured in the yeast one- and two-hybrid assay, respectively (Table I,Table I). Alanine substitution of Gly-141 and Gly-143 (the main chain nitrogen of Gly-141 makes an H2O-mediated contact to a phosphate group and the main chain nitrogen of Gly-143 directly contacts a phosphate) appears to have perturbed the Runt domain fold, as the activity of both proteins was decreased in both the yeast one- and two-hybrid assays. Substitution of the Val-170 side chain with alanine (the main chain nitrogen of Val-170 contacts a phosphate) did not affect either DNA or CBF{beta} binding, at least as measured in the yeast assays.

Substitution of nine residues in the Runt domain that do not directly contact DNA also severely perturbed DNA binding. In most cases (F70A, L75A, W79A, A107G, F146A, and I166A) CBF{beta} heterodimerization was also affected; thus the mutations in all likelihood globally disrupted the Runt domain fold. Three substitutions, R118A, N119A, and I168A, disrupted DNA but not CBF{beta} binding, suggesting that only the local conformation in the vicinity of the substituted residue was affected.

Alanine substitutions at residues that are mutated in human disease in some cases (14 of 27) severely affected DNA binding in the yeast one-hybrid assay (Table I,Table I). These residues include Phe-70, Trp-79, Arg-80, Lys-83, Arg-118, Arg-135, Arg-139, Arg-142, Phe-146, Lys-167, Thr-169, Asp-171, Arg-174, and Arg-177. All but four of these amino acids (Phe-70, Trp-79, Arg-118, and Phe-146) contact DNA. Alanine substitutions of seven residues found mutated in human disease (L62A, V105A, M124A, G138A, S140A, L148A, and I150A) had modest effects on DNA binding, whereas the remainder (H58A, C72A, T154A, and Q158A) had no effect.

Alanine substitutions for residues at the CBF{beta} interface were also examined in the yeast one- and two-hybrid assay (Table I, Table I and Fig. 2C). Only six mutations, W79A, G108A, N109A, F146A, I150A, and T161A, severely affected heterodimerization. The biochemical analysis of the RD(N109A) and RD(T161A) mutant Runt domains is described in the accompanying paper by Zhang et al. (62).

Biochemical Analyses of Runt Domains with Alanine Substitutions in DNA-contacting Residues—To confirm and quantify the yeast one-hybrid results, we purified Runt domain proteins containing alanine substitutions at all 10 residues shown in the crystal structures to make side chain contacts to the DNA (45, 47). We also purified a protein predicted by the yeast one- and two-hybrid assays to have a perturbed structure, the Runt domain F146A mutant. We labeled all the purified mutant Runt domain proteins with 15N and performed two-dimensional heteronuclear single quantum correlation (HSQC) spectroscopy to assess the Runt domain fold (Fig. 3 and Table II). Eight of the alanine-substituted Runt domains that we analyzed yielded spectra similar to that of the wild type Runt domain (Table II). Three mutants, on the other hand, had perturbed structures, including RD(K83A), RD(R135A), and RD(F146A). The perturbed structures of RD(K83A) and RD(R135A) were not predicted because both side chains are relatively solvent-exposed (46.2 and 23.9%, respectively), and both mutant proteins bound CBF{beta} in the yeast two-hybrid assay (Table I,Table I). We characterized these mutants further by performing urea denaturation measurements to assess their thermodynamic stability (Table III). Consistent with the solvent-accessibility data, the urea denaturation data show the stability of RD(K83A) is not altered, whereas RD(R135A) is somewhat destabilized, and RD(F146A) is substantially destabilized. The poor quality of the RD(K83A) HSQC spectrum must arise from some other source, perhaps increased aggregation relative to the wild type Runt domain.



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  		FIG. 3.
15N-1H HSQC spectra of wild type and mutant Runt domain proteins.

 

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  		TABLE II
Summary of 15N-1H HSQC analyses of alanine-substituted RD proteins

 

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  		TABLE III
Structural integrity of alanine-substituted RD as measured by urea denaturation

 

We measured the affinities of the purified Runt domain proteins for DNA in the absence (K2) and presence of CBF{beta} (K4) by EMSA, as described previously (55, 60) (Fig. 4 and Table IV). The alanine substitutions reduced the affinity of the Runt domain for DNA in the range of 10-fold (RD(K83A) and RD(R139A)) to ~1000-fold (R174A) (Table IV).


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  		TABLE IV
Equilibrium binding constants of mutated RD proteins for DNA in the absence (K2) and presence (K4) of CBF{beta}

 

All 10 of the mutant Runt domains with alanine substitutions in DNA-contacting residues produced no detectable {beta}-galactosidase signals in the yeast one-hybrid filter assay (Table I,Table I). However, when we used the somewhat more quantitative liquid assay to assess {beta}-galactosidase activity, we found that both RD(K83A) and RD(R139A) produced more activity than the other eight mutants (45). This is consistent with the biochemical data that showed alanine substitutions of Lys-83 and Arg-139 have the smallest effect on DNA binding. In contrast, the activity of the other alanine mutants (RD(R80A), RD(R135A), RD(R142A), RD(K167A), RD(T169A), RD(D171A), RD(R174A), and RD(R177A)) were indistinguishable and negative in the yeast one-hybrid assay, despite the fact that they have reduced affinities for DNA that range from 24-fold (RD(K167A)) to 1000-fold (RD(R174A)) (Table IV). We conclude that the yeast one-hybrid assay using the HA reporter strain is capable of detecting reductions in DNA binding affinity ranging from approximately 0 to 10-fold relative to the wild type Runt domain.

In the presence of CBF{beta}, the affinities of the same 10 alanine-substituted Runt domains for DNA (K4) were reduced by 27–2470-fold compared with that of the wild type protein (Table IV). CBF{beta} can enhance the affinity of the wild type Runt domain for DNA by ~11–12-fold (K2/K4) (Table IV). Four of the 10 Runt domain mutants including RD(R80A), RD(R135A), RD(K167A), and RD(R177A) have K2/K4 ratios only slightly lower than that of the wild type Runt domain (Table IV); thus CBF{beta} can stimulate DNA binding by the wild type and these four mutant Runt domains to approximately the same extent. In contrast, CBF{beta} enhances the affinities of the Runt domain K83A, R139A, and R174A mutants for DNA by less than 5-fold and of the R142A and T169A mutants by 27–28-fold (Table IV). Thus these mutations make the Runt domain less (K83A, R139A, and R174A) or more (R142A and T169A) sensitive to allosteric regulation by CBF{beta}.

As discussed above, the results we obtained from our yeast one-hybrid assay were basically in agreement with the K2 values we measured by EMSA. To determine whether the yeast one-hybrid assay is also sensitive to differences in K4 values (and thus can detect differences in the affinity of RD-CBF{beta} heterodimers for DNA), we developed a modified yeast one-hybrid assay (Fig. 5A). We co-transformed the yeast reporter strain containing HA Core sites driving lacZ expression with the plasmid encoding the GAL4AD-RD and the plasmid encoding the full-length murine CBF{beta} fused to the GAL4 DNA binding domain (GAL4DBD-CBF{beta}). The results from the modified yeast one-hybrid assay mirrored the trend in the K4 values we determined biochemically. In the presence of CBF{beta}, the {beta}-galactosidase signal from Runt domain mutants RD(K83A), RD(R135A), RD(R139A), RD(R142A), RD(K167A), and RD(R177A) was now within the detectable range of the assay (Fig. 5B). {beta}-Galactosidase activity from mutants with higher K4 values (RD(R80A), RD(T169A), RD(D171A), and RD(R174A)) was not detectable. We also observed the relatively large decrease in K4 for RD(R142A) that was measured by EMSA. The K2 of RD(R142A) is ~1.9 x 10–9 M. If CBF{beta} enhanced the affinity of RD(R142A) for DNA 11-fold, as seen for the wild type Runt domain, the K4 would be ~1.8 x 10–10 M, which should yield only a weak blue or no signal in the modified yeast one-hybrid assay. However, CBF{beta} enhances the affinity of RD(R142A) for DNA by ~27–28-fold (K4 = 7 x 10–11 M), which is within the sensitivity range of the yeast assay, and indeed {beta}-galactosidase activity from RD(R142A) was detected (Fig. 5B).



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  		FIG. 5.
Modified yeast one-hybrid assay. A, schematic diagram of the modified yeast one-hybrid assay. The yeast reporter strain contained the HA core site. B, results from the modified yeast one-hybrid filter assay.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We performed an extensive alanine-scanning mutagenesis study of the Runt domain, targeting 74 amino acids implicated by structural studies to be involved in DNA or CBF{beta} binding and also residues mutated in human disease. We have grouped the alanine mutants that affected DNA binding in a yeast one-hybrid assay into three categories (Table I,Table I and Fig. 2D).

Alanine substitution of 10 amino acids affected DNA binding by disrupting direct contacts to DNA. These mutants severely affected activity in the yeast one-hybrid assay that detects DNA binding but not in the yeast two-hybrid assay that detects the interaction with CBF{beta}.

A second group of mutants perturbed DNA binding but not CBF{beta} binding by potentially disrupting intra-molecular salt bridges or hydrogen bonds that stabilized the RD-DNA complex. Mutant Runt domains within this second category usually showed intermediate levels of {beta}-galactosidase activity in yeast one-hybrid assays but positive results in yeast two-hybrid assays. An example of such a mutation is K144A. Lys-144 in the {beta}E'-F loop makes potential salt bridges to the side chains of Asp-110 and Glu-111 in loop {beta}C-D, thus bridging the {beta}E'-F loop and the {beta}C-D loop. The K144A substitution will disrupt these salt bridges (45). Another example is Ser-140, which forms potential hydrogen bonds to Lys-144 and Asn-109 and serves to bridge the Runt domain {beta}C-D loop, {beta}E'-F loop, and the parallel {beta}A'-G' sheet. The {beta}A'-G' sheet in turn makes base-specific contacts to the DNA. The S140A substitution should disrupt the formation of hydrogen bonds to Lys-144 and Asn-109 and ultimately destabilize the {beta}A'-G' sheet (45). Yet another example is Arg-118 in strand {beta}D, which forms a hydrogen bond with the side chain of Asn-82 in the {beta}A'-B loop, that may serve to stabilize the DNA-contacting residue Lys-83 or perhaps the entire {beta}A'-B loop.

A third group of mutations in all likelihood affected DNA binding by disrupting the overall fold of the Runt domain. Mutants in this category usually have reduced {beta}-galactosidase activities in both yeast one- and two-hybrid assays. Most of these amino acids are nonpolar residues whose side chains are buried in the hydrophobic core of the protein, such as Trp-79 and Phe-146. A subcategory within the group of mutations that perturb the fold are those that might cause local rather than global changes in conformation that would affect DNA binding. For example, Gly-141 and Gly-143 in the {beta}E'-F loop flank the important DNA-contacting residue Arg-142. Gly-141 and Gly-143 use a backbone NH to contact DNA and are quite distant from the CBF{beta}-binding surface. In both cases, the backbone {phi} and {Psi} angles adopted by these residues are highly unfavorable for any other amino acid, and substitutions are likely to cause a significant distortion of the {beta}E'-F loop conformation. The {beta}E'-F loop adopts two different conformations in the absence and presence of CBF{beta} and DNA in the crystal structures (42, 43, 45, 47, 48). The high conformational flexibility of Gly-141 and Gly-143 may be critical in providing for the flexibility of the {beta}E'-F loop and its response to allosteric regulation by CBF{beta} and DNA. Another potential example of a mutation that might cause a local conformational change is I168A. Ile-168 is located in the {beta}G' strand, immediately adjacent to the C-terminal tail of the Runt domain that docks in the major groove of the DNA (Fig. 2B). Ile-168 is flanked on either side by two DNA-contacting residues Lys-167 and Thr-169. The I168A mutation, which affects activity in the yeast one-hybrid but not in the yeast two-hybrid assay, may perturb the orientation of the Lys-167 and Thr-169 side chains. We should note though that perturbed activity in the yeast one- and two-hybrid assays is not necessarily a reliable indication of protein stability. For example, two mutants with decreased activity as measured in these two assays, RD(D66A) and RD(T161A), had a normal Runt domain fold as determined by 15N-1H HSQC spectroscopy (62).

The DNA binding activity of the Runt domain appears to be extremely sensitive to amino acid substitutions. Almost two-thirds of the 74 alanine mutants we tested in our yeast one-hybrid assay affected DNA binding to some extent (Table I,Table I and Fig. 2B). The DNA-binding domain of another protein with an s-type immunoglobulin fold, p53, displays a similar sensitivity to mutations that lead to loss of function (63, 64). The tumor-derived mutants of p53 fall into two distinct categories. The large majority of the mutations affect amino acids that are critical for maintaining the fold of the highly conserved DNA-binding core domain, and these mutants are referred to as "structural mutants." These structural mutants affect DNA binding by causing defects ranging from partial to global destabilization and unfolding of the p53 protein (63, 65–67). Other mutations are the so-called "functional mutants" that affect residues directly involved in protein-DNA interactions (63, 67, 68). Only 10 mutants of the Runt domain, for which we have measured DNA binding constants (Table IV), can be considered functional mutants. As is the case for p53, most of the alanine mutants we tested that have reduced affinities for DNA binding can be considered structural mutants. These mutations affect DNA binding either by disrupting the fold of the protein globally (e.g. W79A and F146A) or locally (R118A, S140A, K144A, and I168A). There is also a group of structural mutants involving residues in the areas of the Runt domain that adopt different conformations in the free Runt domain versus the Runt domain in binary and ternary complexes (42, 43, 45, 47, 60). These regions include the {beta}C-D loop (Asn-109 to Ala-115), the {beta}E'-F loop (Gly-138 to Leu-148), and the "S-switch" region that encompasses the C-terminal end of the {beta}G' strand (Tyr-162 to His-163), the {beta}G'-G loop (Arg-164 to Ala-165), and the N-terminal residues of {beta}G (Ile-166 to Lys-167). CBF{beta} is thought to increase the DNA binding affinity of the Runt domain by stabilizing a single conformation of these flexible regions. Mutations in the flexible regions may affect DNA binding by perturbing the structure locally and perhaps locking the Runt domain into a conformation that renders it less sensitive to allosteric regulation by CBF{beta} or DNA. This type of structural mutant has not been described for p53.

In p53, the most frequent missense mutations associated with cancer affect six "hot spot" residues (Arg-175, Gly-245, Arg-248, Arg-249, Arg-273, and Arg-282) (63). Among these six hot spot residues five are arginines, with Arg-248 and Arg-273 making direct DNA contacts. The remaining four hot spot residues that do not contact DNA directly play a critical role in stabilizing the p53 fold. In the Runt domain, Arg-174 is the most frequently mutated residue in human patients and therefore can also be considered a mutation hot spot residue (Table I,Table I). This correlates with our equilibrium binding constant measurements that determined Arg-174 is the residue that contributes the greatest energy to DNA binding. In addition to Arg-174, other frequently mutated residues in RUNX1 or RUNX2 found in human disease include Lys-83, Met-124, Arg-139, Ser-140, Asp-171, and Arg-177 (Table I,Table I). Of these, Lys-83, Arg-139, Asp-171, and Arg-177 directly contact DNA, and alanine substitutions reduce the affinities of the Runt domain for DNA by ~10- (K83A and R139A) to 200-fold (D171A) (Table IV). The Runt domain and p53 both use an arginine residue (Arg-248 for p53 and Arg-142 for Runt domain) to make contacts in the minor groove of the DNA. In p53, Arg-248 is the most frequently mutated hot spot residue (9.6% of all p53 mutations) (63). However, mutations involving Arg-142 of the Runt domain are rare. So far only one mutation, R142C, has been reported in CCD patients (40).

CBF{beta} can enhance the affinities of both RD(R142A) and RD(T169A) for DNA by about 27-fold, compared with the 11–12-fold enhancement seen for the wild type Runt domain (Table IV). The Runt domain and DNA each induce conformational changes in the other molecule to achieve optimal binding (43, 45, 47, 60, 61). The free DNA-binding site is bent by ~16°, and the Runt domain bends the DNA an additional 7–8° toward the protein (43). One hypothesis for the increased sensitivity of RD(R142A) and RD(T169A) to allosteric regulation by CBF{beta} is that in the absence of the CBF{beta} subunit, Arg-142 and Thr-169 may be responsible for mediating most of the conformational change in DNA, whereas in the presence of CBF{beta}, more amino acids may contribute to maintaining the optimal DNA conformation, rendering the individual contributions by Arg-142 and Thr-169 less important. If this were the case, we should see significantly reduced levels of DNA bending induced by the RD(R142A) and RD(T169A) proteins. However, both RD(R142A) and RD(T169A) bent DNA to the same degree as the wild type Runt domain in EMSA-based bending assays (69, 70) (data not shown). A second hypothesis is that because DNA can also induce a conformational change in the Runt domain, it is possible that both Arg-142 and Thr-169 are responsible for mediating most of this change in the absence of CBF{beta}. Specifically, Arg-142 is in the center of the {beta}E'-F loop and reaches into the minor groove of the DNA. Arg-142 also contacts the phosphate backbone of DNA together with Arg-139, Gly-141, and Gly-143 (45, 47). In the absence of CBF{beta}, substitution of the Arg-142 side chain with that of alanine might somehow block or destabilize the DNA-induced structural rearrangement of this flexible region. The same could also be true for Thr-169, which lies between the C-terminal tail (which is important for determining the specificity of DNA recognition) and the S-switch region (amino acids Tyr-162 to Lys-167) in the crystal structure. In the presence of CBF{beta}, perhaps more amino acids contribute to mediating the DNA-induced conformational rearrangement, so that the individual contributions by Arg-142 and Thr-169 become less important. Currently there are no experimental data to support or refute this hypothesis.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants AI39536, AI01481, and AI45120, the Leukemia and Lymphoma Society (to J. H. B.), and National Institutes of Health Grants CA058343 [GenBank] , CA89419, and CA75611 (to N. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by National Institutes of Health Grant T32 GM08704. Back

** Present address: Structural Biology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, E-28029 Madrid, Spain. Back

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 603-650-1159; Fax: 603-650-1128; E-mail: nancy.speck{at}dartmouth.edu.

1 The abbreviations used are: CBFs, core-binding factors; RD, Runt domain; EMSAs, electrophoretic mobility shift assays; AML, acute myeloid leukemia; MDS, myelodysplasias; FPD, familial platelet disorder; CCD, cleidocranial dysplasia; MLV, murine leukemia virus; HA, high affinity; HSQC, heteronuclear single quantum correlation. Back


   ACKNOWLEDGMENTS
 
We thank Xin Zhao, Lina Zhang, Jing Zhang, and Amy Davis for their assistance in making the Runt domain alanine substitutions. We thank Alok Pant for help in performing some of the yeast two-hybrid assays and Camille Shammas for help in generating the structure images. We also thank Yen-Yee Tang for technical help and valuable discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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