Structure and function in c-Myc and Grx4 : two key proteins involved in transcriptional activation and oxidative stress

The proto-oncogene c-myc is critical for growth and development and deregulation of c-myc expression affects the initiation and expansion of a wide range of human cancers, many of them aggressive. cMyc is a multi-domain protein, where it's C-terminal basic helix-loop-helix leucine zipper (bHLH-Zip) domain heterodimerizes with Max to accomplish gene regulatory activity. A biophysical investigation of the biological important regions flanking the bHLH-Zip of Max found that they promote increased folding of Max and play a regulatory role in determining the affinity to DNA. The activity of the N-terminal c-Myc transactivation domain (TAD) is regulated by binding to a range of proteins. A molecular mapping of c-Myc TAD regulatory activities has utmost relevance as a molecular drug would contribute substantially to cancer treatment. An extended c-Myc TAD, MYC1-167, includes the unstructured c-Myc TAD (aa 1-143) together with a C-terminal segment, which were found to promote folding. Surprisingly, Myc1-167 displays the characteristics of a helical molten globule, and binds both to Myc Modulator-1 (MM-1) and TATA box-binding protein (TBP). Although its C-terminal region (MYC92-167) has a partly helical fold and binds both MM-1 and TBP, neither N- nor C-terminal regions Of MYC1-167 bind target proteins with as high affinity as the entire MYC1-167, or display molten globule properties. A screen of common interaction patches in c-Myc TAD binding proteins resulted in identification of a peptide binding motif in TBP which interacts with c-Myc TAD and reduces cell growth in cMyc overexpressing cells. Structural mapping of interacting residues in the TBP derived peptide by NMR and Biacore lead to the identification of a peptide segment belonging to the same binding motif in the histone acetyltransferase complex member TRRAP, which bound c-Myc TAD with even higher affinity. The location of a common c-Myc interacting patch in TBP and TRRAP proposes a mechanism for recruitment of c-Myc to the preinitiation complex and suggests novel routes for therapeutic strategies. Glutaredoxins are ubiquitous proteins found in most living organisms and they function by performing redox regulation in the cell. Despite the wealth of information regarding glutaredoxins employing a dithiol mechanism for the reduction of their substrates, little is known about the monothiol glutaredoxins. E. coli Glutaredoxin 4 (Grx4), which showed no activity in the classical glutaredoxin (HED) assays, could still be oxidized by glutathione and form an internal disulfide. This internal Grx4 disulfide was a direct substrate for NADPH and E. coli Thioredoxin reductase, while the mixed disulfide was reduced by E. coli Glutaredoxin 1, suggesting extremely narrow substrate specificity with regulatory implications. The three-dimensional structure of reduced Grx4 was determined by NMR and comprises a glutaredoxin-like alpha-beta fold with stringently conserved structural features, likely to be present in all monothiol glutaredoxins. The absence of classical glutaredoxin activity in E. coli Grx4 is understood based on small but significant structural differences in the glutathione binding region, and through the lack of a conserved second GSH binding site. MALDI experiments suggest that disulfide formation on glutathionylation is accompanied by significant structural changes, and together with detailed structural evaluation suggests that the disulfide-forming region forms an active site