Forced homodimerization of the c-Fos leucine zipper in designed bHLHZ-like hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos.
Although the c-Fos leucine zipper (LZ) does not form a homodimer in its native basic region/leucine zipper (bZIP) structure, we found that it is capable of homodimerization and promoting protein folding in engineered basic region/helix-loop-helix/leucine zipper (bHLHZ) hybrid proteins MaxbHLH-Fos and ArntbHLH-Fos, in which the bHLH subdomains of Max and Arnt are fused to the c-Fos LZ. By using the in vivo yeast one-hybrid system and in vitro circular dichroism and quantitative fluorescence anisotropy, we demonstrated that attachment of the c-Fos LZ to the otherwise unstructured MaxbHLH resulted in a hybrid bHLHZ-like protein now competent for homodimerization and DNA binding at the cognate E-box site, CACGTG. In ArntbHLH-Fos, the c-Fos LZ promoted proper folding of the HLH structure, although unlike MaxbHLH, ArntbHLH alone is capable of homodimerization and DNA binding. In addition, by comparing the E-box binding and secondary structures of MaxbHLH-Fos and two derivatives containing targeted mutations in the c-Fos LZ, we found that cooperative communication exists between the bHLH and LZ: proper folding of the four-helix bundle in the HLH region could be induced by the c-Fos LZ, and the HLH dimerization region could force homodimerization of the c-Fos LZ. These results demonstrate that although intrinsically unfavorable, the c-Fos LZ can homodimerize, demonstrating that the same c-Fos LZ element can yield orthogonal differences in structure and/or DNA-binding function within different transcription factor families, including the bZIP and bHLHZ.
Department of Chemistry, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
Gang
G
Chen
Antonia T
AT
De Jong
Jumi A
JA
Shin
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D019281:Dimerization; D024721:E-Box Elements; D019077:Helix-Turn-Helix Motifs; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins