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Chloride binding by the AML1/Runx1 transcription factor studied by NMR
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
Faculty of Medicine, Molecular Biology (Faculty of Medicine).
Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
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2001 (English)In: FEBS Letters, ISSN 0014-5793, Vol. 488, no 1-2, 81-4 p.Article in journal (Refereed) Published
Abstract [en]

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 (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,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.

Place, publisher, year, edition, pages
2001. Vol. 488, no 1-2, 81-4 p.
Keyword [en]
AML1, Runt domain, 35Cl, Nuclear magnetic resonance spectroscopy, Anion binding to protein
National Category
Structural Biology
URN: urn:nbn:se:umu:diva-17923DOI: doi:10.1016/S0014-5793(00)02390-5PubMedID: 11163800OAI: diva2:157596
Available from: 2008-05-05 Created: 2008-05-05 Last updated: 2012-06-28Bibliographically approved
In thesis
1. The hematopoietic transcription factor RUNX1: a structural view
Open this publication in new window or tab >>The hematopoietic transcription factor RUNX1: a structural view
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.

The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.

The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.

During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.

The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution

synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet), 2004. 29 p.
Molecular medicine, RUNX1, Runt domain, CBFβ, transcription factor, leukaemia, protein crystallography, anomalous diffraction, Molekylärmedicin
National Category
Medical Genetics
Research subject
Molecular Biology
urn:nbn:se:umu:diva-192 (URN)91-7305-476-3 (ISBN)
Public defence
2003-09-05, Umeå, 10:00
Available from: 2004-02-06 Created: 2004-02-06 Last updated: 2012-06-28Bibliographically approved

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Wolf-Watz, MagnusBäckström, StefanGrundström, ThomasSauer, Uwe
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