Hisayuki Morii

Mechanism of c-Myb–C/EBPβ Cooperation from Separated Sites on a Promoter

c-Myb, but not avian myeloblastosis virus (AMV) v-Myb, cooperates with C/EBPβ to regulate transcription of myeloid-specific genes. To assess the structural basis for that difference, we determined the crystal structures of complexes comprised of the c-Myb or AMV v-Myb DNA-binding domain (DBD), the C/EBPβ DBD, and a promoter DNA fragment. Within the c-Myb complex, a DNA-bound C/EBPβ interacts with R2 of c-Myb bound to a different DNA fragment; point mutations in v-Myb R2 eliminate such interaction within the v-Myb complex. GST pull-down assays, luciferase trans-activation assays, and atomic force microscopy confirmed that the interaction of c-Myb and C/EBPβ observed in crystal mimics their long range interaction on the promoter, which is accompanied by intervening DNA looping.

Multi-state thermal transitions of proteins - DNA-binding domain of the c-Myb oncoprotein

The DNA binding domain of c-Myb protein, a regulator of the transcription, consists of three homologous tandem repeats (Rl, R2 and R3) with 51-52 amino acid residues. Previously, we found that the thermal transition of the binding domain (RlR2R3) is three-state transition and that the intermediate state is related to the lower stability of R2 than the other two. In order to clarify the thermodynamic characteristics of the most unstable R2, we synthesized two mutants of R2, R2(V103I) and R2(V103L), and examined their thermodynamic properties by circular dichroism and differential scanning calorimetry. The thermal transition temperature (T,,) and enthalpy change (AH) obtained by CD for R2(V103I) and R2(V103L) were 47.1 and 62.6 C, and 144 and 166 kJ mol, respectively. The corresponding values by DSC were 47.5 and 62.8 T, and 140.1 and 173.4 kJ mol, respectively. Especially, the values of T,, and AH for a(V103L) by CD (DSC) are significantly higher than those for R2, which are 43.7 (43.8) C and 125.4 (133.6) kJ mol. The larger stabilizing effect by the substitution was found for Leu mutant. The results suggest that not only the cavity effect but also the contributions of conformational change in native and denatured states, and of the interactions of the side chains with water should be taken into account. binding domain of c-Myb protein, a transcriptional regulatory factor, consists of three homologous tandem repeats (Rl, R2 and R3) with 51-52 amino acid residues (3). The homologies between R1 and R2, R2 and R3, and R3 and R1 are 46%, 31%, and 33%, respectively. The solution structures of the three repeats have been determined by NMR analysis (4, 5). Each repeat has conformation similar to helix-turn-helix motif with three well-defined helices. Three conservative tryptophans in each repeat participate in forming a hydrophobic core, which definitely characterizes the structure of the repeat. Comparison of the three repeat structures indicated that there existed a cavity in the hydrophobic core of R2. Val is located at the position 103 in the frst helix of R2, whereas the corresponding amino acids in R1 and R3 are more bulky Leu 51 and Ile155, respectively (5). The presence of the cavity in R2 was found to cause conformational flexibility of R2 and play an important role in binding and recognition of DNA (5, 6). The cavity also decreases the conformational stability of R2 compared with R1 and R3, which gives rise to the three-state thermal transition of RlR2R3 (7) and R2R3 (6). The mutant of R2R3 in which Val 103 in R2 is replaced to Leu showed higher thermal stability than wild-type R2R3 (6). Both wild type and the mutant of R2R3 were