Biomedical subjects

D Meierhans

Publications and source records attributed to D Meierhans.

6 recordsLinked to original sources

PubMed1999

Acquisition of myogenic specificity through replacement of one amino acid of MASH-1 and introduction of an additional alpha-helical turn.

The homologous transcription factors Myf-5, MyoD, myogenin, MRF-4, and MASH-1 bind with high affinity and modest sequence specificity to DNA containing an E-box (CANNTG). This similarity of the in vitro DNA binding specificity is in sharp contrast to the high physiological specificity displayed by these proteins. Myf-5, MyoD, myogenin, and MRF-4 induce cells to differentiate along a myogenic pathway, while MASH-1 promotes the differentiation of neuronal precursor cells. We show here that MASH-1 can be converted into a protein capable of inducing myogenesis in fibroblasts by replacing leucine (130) of MASH-1 with lysine and introducing an additional turn into its basic recognition helix. These changes do not significantly alter the DNA binding properties of the proteins in cell free conditions. Crystallographic data for the DNA complexes of MyoD and E12 suggest that Leu (130) points away from the DNA into the solvent. We postulate that the identity of the amino acid in position 130 is important for protein-protein interactions that might affect the DNA binding specificities displayed by BHLH-proteins in vivo and form the molecular basis of the different physiological properties of the myogenic and neurogenic BHLH-proteins.

Amino Acid Sequence↗

PubMed1998

The N-terminal methionine is a major determinant of the DNA binding specificity of MEF-2C.

Members of the MEF-2 family of transcriptional regulators positively modulate the activity of basic helix-loop-helix proteins in both myogenic and neurogenic cell lineages. Previous work had shown that MEF-2C(2-117), a protein fragment comprising the dimerization and DNA-binding domains of MEF-2C but lacking the N-terminal methionine, bound to AT-rich DNA sequences with high affinity. MEF-2C(2-117) did not discriminate between different AT-rich sequences. We now report the in vitro DNA binding properties of a MEF-2C fragment containing the N-terminal methionine. Measurements of the apparent dissociation constants of the complexes of GG-MEF-2C(1-117) revealed that different AT-rich sequences are bound with different affinities; in particular MEF site containing DNA (CTATAAATAG) is bound preferentially to DNA containing a SRF site (CATAAATG). Strikingly, when the shorter AT run consisted of six alternating thymines and adenines, almost wild-type affinity was observed. Irrespective of the particular DNA sequence, all circular dichroism spectra of the DNA complexes of GG-MEF-2C(1-117) were superimposable and characterized by an identical maximal ellipticity at 269.5 nm, suggesting similar DNA conformations. Bending analysis by circular permutation assay revealed that on complex formation MEF-2C(2-117) induced cognate DNA to bend by 49 degrees, while heterologous DNA remained unbent. In the presence of the N-terminal methionine, however, all DNA sequences were bent by 70 degrees. The above results suggest an important function for the N-terminal methionine in properly orientating MEF-2C on the DNA.

Amino Acid Sequence↗

PubMed1998

Thermodynamics of the DNA binding reaction of transcription factor MASH-1.

MASH-1, a member of the basic helix-loop-helix (BHLH) family of transcription factors, promotes the differentiation of committed neuronal precursor cells. We have determined the thermodynamic parameters of the DNA binding reaction of the BHLH domain of MASH-1 (MASH-BHLH) by isothermal titration calorimetry and found that the specificity of the binding reaction was rather low. At 27 degrees C, the association constant for binding was 5.13 (+/-0.51) x 10(8) M-1 for an E-box containing oligonucleotide, while for a heterologous DNA sequence it was 5.14 (+/-1.93) x 10(7) M-1. The reaction enthalpy and the reaction entropy were strongly dependent on the temperature, but the reaction free energy was almost independent of temperature. The association reaction was enthalpically driven throughout the physiological temperature range and characterized by a large negative heat capacity change. No change in the protonation state of the protein and/or the DNA was observed at pH 6. Within experimental error, the reaction was independent of pH between pH 6 and 8. Dissection of the entropy change of the binding reaction indicated that binding was coupled to local protein folding of approximately 25 amino acids per protein subunit. The circular dichroism spectra of free and DNA-bound MASH-BHLH revealed the formation of additional alpha-helical structure comprising approximately 25 amino acids upon complex formation. Therefore, while the basic region was in an alpha-helical conformation in the DNA complex, in free MASH-BHLH it was substantially unfolded even at concentrations where the protein is mainly dimeric. The association between MASH-1 and DNA is therefore an example of "induced fit".

Amino Acid Sequence↗

PubMed1997

High affinity binding of MEF-2C correlates with DNA bending.

To regulate lineage-specific gene expression in many cell types, members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors cooperate with basic helix-loop-helix (bHLH) proteins, which show only limited intrinsic DNA binding specificity. We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural characteristics recognized by MEF-2C. Measurements of the apparent dissociation constants of MEF-2C complexes with several DNA sequences revealed that MEF-2C bound with high affinity to DNA sequences containing a MEF site. Mutations in the MEF site which did not affect the bendability of the DNA changed the free energy of binding only marginally. However, reducing the intrinsic bendability of the DNA binding site through an AA-->GC substitution increased the half-maximal binding concentration of MEF-2C by almost one order of magnitude. Electrophoretic mobility shift assays revealed markedly reduced MEF-2C binding to DNA containing 2,6-diaminopurine. On binding to MEF-2C the maximum ellipticity at 275 nm in the CD spectrum of DNA containing a MEF site was red shifted by 4 nm and its intensity reduced significantly, while a slight blue shift of <1 nm was observed for a mutant DNA sequence with reduced bendability (AA-->GC). Bending analysis by circular permutation assay revealed that the DNA in the cognate complex was bent by 49 degrees , while the DNA in the complex with the mutant oligonucleotide was largely unbent.

2-Aminopurine↗

PubMed1997

High level expression in soluble form, one step purification, and characterization of the DNA binding domain of MEF-2C.

Members of the MEF-2 family of transcription factors act as coregulators of basic helix-loop-helix (BHLH) proteins in the control of lineage specific gene expression in many cell types through direct interaction between the respective DNA binding domains. To make possible a thorough biochemical, biophysical, and structural characterization of the properties of myocyte enhancer factor (MEF) proteins and of their interactions with BHLH-proteins, a simple system for high level expression and rapid purification of myocyte enhancer factor-2C (MEF-2C) was developed. A T7 expression system was used to produce in high yield in Escherichia coli an N-terminal fragment of MEF-2C comprising both the MADS box and the MEF domain. Purification by a single round of cation-exchange chromatography on a Resource-S HPLC column at elevated pH afforded an essentially pure protein. Recombinant MEF-2C (1-117) bound with high affinity to the MEF consensus DNA binding site (CTATAAATAG). Mutations in this sequence that replaced adenines with thymine or vice versa did not significantly alter the affinity for MEF-2C(1-117). The introduction of G-C pairs into the core of the MEF-site, however, dramatically increased the concentration of MEF-2C(1-117) needed for half maximal DNA binding. We propose an explanation of the DNA binding specificity of MEF-2C based on the intrinsic bending properties of the unbound DNA.

Amino Acid Sequence↗

PubMed1996

Basic helix-loop-helix protein MyoD displays modest DNA binding specificity.

The expression of MyoD can activate muscle specific genes and myogenic differentiation in many cell types. The hypothesis that the DNA binding specificity of MyoD is responsible for its biological specificity was tested. Homodimers of MyoD bind to E-box containing DNA with high affinity, but do not form stable and well defined complexes with heterologous DNA sequences. The physiologically active heterodimer of MyoD and E12 binds an oligonucleotide containing an E-box sequence with an affinity only two orders of magnitude higher than a completely unrelated DNA sequence, stressing the importance of cooperative interactions with other proteins of the transcriptional machinery for specific gene activation.

Amino Acid Sequence↗