The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor β, CBFβ, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25Å resolution and compare its conformation to previously published structures in complex with DNA, CBFβ or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFβ-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFβ alone; suggesting that one role of CBFβ is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl− ions. In this paper we have determined Cl− binding affinities of AML1 by 35Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl− with a dissociation constant (Kd,Cl) of 34 mM. If CBFβ is added to form a 1:1 complex, the Kd,Cl value increases to 56 mM. Homology modeling suggests that a high occupancy Cl− binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl− ion. Possible biological roles of Cl− binding are discussed based on these findings.

The acute myeloid leukaemia 1 (AML1) protein belongs to the Runx family of transcription factors and is crucial for haematopoietic development. The genes encoding Runx1 and its associated factor CBF beta are the most frequent targets for chromosomal rearrangements in acute human leukaemias. In addition, point mutations of Runx1 in acute leukaemias and in the familial platelet disorder FPD/AML cluster within the evolutionary conserved runt domain that binds both DNA and CBF beta. Here, the crystallization of the Runx1 runt domain is reported. Crystals belong to space groups C2 and R32 and diffract to 1.7 and 2.0 A resolution, respectively.

Crystallographic studies of thiamin-diphosphate-dependent transketolase from Saccharomyces cerevisiae suggested the invariant active-site residue H103 as a possible enzymic group binding the C1 hydroxyl group of the donor substrate and stabilizing the reaction intermediate. To test this hypothesis, H103 was replaced by alanine, asparagine and phenylalanine using site-directed mutagenesis. The crystallographic analysis of the mutant transketolases verified that no structural changes occurred as a consequence of the side-chain replacements. The residual catalytic activities of the mutant enzymes were 4.3% for the H103A, 2.4% for the H103N and 0.1% for the H103F mutant transketolase. Further kinetic analysis of the H103A and H103N mutant enzymes showed that the Km values for the coenzyme were increased by about eightfold. The Km values for the acceptor substrate ribose 5-phosphate were similar to the Km value for wild-type transketolase. However, the Km value for the donor substrate, xylulose 5-phosphate is increased more than tenfold in these two mutants. Circular dichroism spectra of the mutant enzymes also indicated a weaker binding of the donor substrate and/or a less stable reaction intermediate. These observations provide further evidence in support of the proposed role for this invariant residue in recognition of the donor substrate by forming a hydrogen bond between the side chain of H103 and the C1 hydroxyl group of the sugar phosphate. The significant decrease in catalytic activity suggests that this residue also facilitates catalysis, possibly by maintaining the optimal orientation of the donor substrate and reaction intermediates.