Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module |
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Authors: | Riley, L.G. Ralston, G.B. Weiss, A.S. |
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Affiliation: | Department of Biochemistry, University of Sydney NSW 2006, Australia |
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Abstract: | Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDSPAGE snapshot analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZGST::rFosLZGSTheterodimers whereas rJunLZGST::rFosLZ and rJunLZ::rFosLZGSTformed readily. Furthermore, rJunLZGST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (Ka >106 M-1) with no higher multimeric forms. rFosLZGST weaklyassociates beyond a dimer (Ka {small tilde}4x105 M-1) and rJunLZGSTassociates indefinitely (Ka {small tilde}4x106 M-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired. |
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Keywords: | c-Fos/ c-Jun/ glutathione S-transferase/ leucine zipper/ multimer |
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