The importance of the linker connecting the repeats of the c-Myb oncoprotein may be due to a positioning function.
The DNA-binding domain of the oncoprotein c-Myb consists of three imperfect tryptophan-rich repeats, R1, R2 and R3. Each repeat forms an independent mini-domain with a helix-turn-helix related motif and they are connected by linkers containing highly conserved residues. The location of the linker between two DNA-binding units suggests a function analogous to a dimerisation motif with a critical role in positioning the recognition helices of each mini-domain. Mutational analysis of the minimal DNA-binding domain of chicken c-Myb (R2 and R3), revealed that besides the recognition helices of each repeat, the linker connecting them was of critical importance in maintaining specific DNA-binding. A comparison of several linker sequences from different Myb proteins revealed a highly conserved motif of four amino acids in the first half of the linker: LNPE (L138 to E141 in chicken c-Myb R2R3). Substitution of residues within this sequence led to reduced stability of protein-DNA complexes and even loss of DNA-binding. The two most affected mutants showed increased accessibility to proteases, and fluorescence emission spectra and quenching experiments revealed greater average exposure of tryptophans which suggests changes in conformation of the proteins. From the structure of R2R3 we propose that the LNPE motif provides two functions: anchorage to the first repeat (through L) and determination of the direction of the bridge to the next repeat (through P).
Hegvold
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Department of Biochemistry, University of Oslo, Norway.
Gabrielsen
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OS
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019077:Helix-Turn-Helix Motifs; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011089:Polydeoxyribonucleotides / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb; D012091:Repetitive Sequences, Nucleic Acid; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D000178:Acrylamides; D015342:DNA Probes; D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D014157:Transcription Factors; D020106:Acrylamide; C031156:poly d(I-C); D014364:Tryptophan; D002918:Chymotrypsin; D014357:Trypsin