Structure of the B-Myb DNA-binding domain in solution and evidence for multiple conformations in the region of repeat-2 involved in DNA binding: implications for sequence-specific DNA binding by Myb proteins.
A range of double and triple resonance heteronuclear NMR has been used to obtain nearly complete sequence-specific 15N, 13C and 1H resonance assignments for a 110-residue protein corresponding to the B-Myb DNA-binding domain (B-MybR2R3) and to determine its secondary structure in solution. The protein was found to contain two stable helices in repeat-2 (R2) and three in repeat-3 (R3), involving residues K12-K24 (R2-1), W30-H36 (R2-2), E64-V76 (R3-1), W81-L87 (R3-2) and D93-K105 (R3-3). In addition, the chemical shift and nuclear Overhauser effect data suggest that amino acids Q44-W49 near the C-terminus of R2 form an unstable or nascent helix, which could be stabilised on binding to a specific DNA target site. The two N-terminal helices in R2 and R3 occupy essentially identical positions in the two domains, consistent with the high level of sequence similarity between these regions. In contrast, the C-terminal region forming the third helix in R3 shows low sequence similarity with R2, accounting for the differences in secondary structure. In the case of B-MybR2R3, there is a clear chemical shift and line-broadening evidence for the existence of multiple conformations in the C-terminal region of R2, which is believed to form one half of the DNA-binding site. We propose that conformational instability of part of the DNA-binding motif is a way of increasing the specificity of Myb proteins for a relatively short (6-bp) DNA target site by reducing their affinity for non-specific DNA sequences compared to specific sites.
Carr
M D
MD
Laboratory of Molecular Structure, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, UK.
Wollborn
U
U
McIntosh
P B
PB
Frenkiel
T A
TA
McCormick
J E
JE
Bauer
C J
CJ
Klempnauer
K H
KH
Feeney
J
J
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D012996:Solutions; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA