Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module.
Human c-Jun and c-Fos leucine zipper domains were examined for their ability to serve as autonomous dimerization domains as part of a heterologous protein construct. Schistosoma japonicum glutathione S-transferase (GST) was fused to recombinant Jun leucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains. SDS-PAGE 'snapshot' analyses based on disulphide linkage of monomers demonstrated the ability of rJunLZ to function as a dimerization motif in a foreign protein environment. Steric hindrance prevented formation of rJunLZ-GST::rFosLZ-GST heterodimers whereas rJunLZ-GST::rFosLZ and rJunLZ:: rFosLZGST formed readily. Furthermore, rJunLZGST generated homodimers suggesting fusion protein heterodimers interact differently to homodimers. Gel filtration chromatography confirmed that GST is a dimer in solution and that attachment of a leucine zipper domain allows further interactions to take place. Sedimentation equilibrium analyses showed that GST is a stable dimer (K(a) > 10(6) M(-1)) with no higher multimeric forms. rFosLZ-GST weakly associates beyond a dimer (K(a) approximately 4 x 10(4) M(-1)) and rJunLZ-GST associates indefinitely (K(a) approximately 4 x 10(5) M(-1)) [corrected], consistent with an isodesmic model of association. The interaction of these leucine zippers independently of GST association demonstrates their utility in the modification of proteins when multimer formation is desired.
Riley
L G
LG
Department of Biochemistry, University of Sydney, NSW, Australia.
Ralston
G B
GB
Weiss
A S
AS
D000818:Animals; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016350:Leucine Zippers; D008958:Models, Molecular; D011487:Protein Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D012549:Schistosoma japonicum / Q000201:enzymology; D013917:Thrombin / Q000378:metabolism; D014461:Ultracentrifugation
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D013917:Thrombin