Determinants of helix-loop-helix dimerization affinity. Random mutational analysis of SCL/tal.
Dimerization represents a key regulatory step in the function of basic helix-loop-helix transcriptional factors. In many instances tissue-specific basic helix-loop-helix proteins, such as the hematopoietic factor SCL/tal or the myogenic factor MyoD, interact with ubiquitously expressed basic helix-loop-helix proteins, such as E2A or E2-2. Such dimerization is necessary for high affinity, sequence-specific DNA binding. Previous biochemical and structural studies have shown the helix-loop-helix region to be necessary and sufficient for this interaction. In the present study, we analyzed the relative affinities of various helix-loop-helix interactions using the yeast two-hybrid system. The relative affinities of selected helix-loop-helix species for the partner protein E2-2 were as follows: Id2 > MyoD > SCL/tal. Mutants of SCL/tal with increased affinity for E2-2 were selected from a library of randomly mutated basic helix-loop-helix domains. The amino acid changes in these high affinity versions of SCL/tal introduced residues that resembled those in the corresponding positions of the Id proteins and MyoD. One of the mutants, SCL 12, also contained mutations in highly conserved residues previously thought to be necessary for dimerization. This mutant of SCL demonstrated diminished temperature sensitivity in in vitro interaction assays as compared with the wild type protein. Computational modeling of helix-loop-helix dimers provides an explanation for the increased dimerization affinity of SCL mutant 12.
Goldfarb
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AN
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. ang@po.cwru.edu
Lewandowska
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Shoham
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M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017570:MyoD Protein / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors
D001704:Biopolymers; D004268:DNA-Binding Proteins; D017570:MyoD Protein; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors