Interfacial asparagine residues within an amide tetrad contribute to Max helix-loop-helix leucine zipper homodimer stability

The transcription factor Max is the obligate dimerization partner of the My c oncoprotein. The pivotal role of Max within the Myc regulatory network is dependent upon its ability to dimerize via the helix-loop-helix leucine zi pper domain. The Max homodimer contains a tetrad of polar residues at the i nterface of the leucine zipper domain. A conserved interfacial Asn residue at an equivalent position in two other leucine zipper proteins has been sho wn to decrease homodimer stability. The unusual arrangement of this Gln-Asn /Gln'-Asn' tetrad prompted us to investigate whether Asn(92) plays a simila r role in destabilizing the Max homodimer. This residue was sequentially re placed with aliphatic and charged residues. Thermal denaturation, redox tim e course and analytical ultracentrifugation studies show that the N92V muta tion does not increase homodimer stability. Replacing this residue with neg atively charged side chains in N92D and N92E destabilizes the mutant homodi mer. Further replacement of Gln(91) indicated that H bonding between Gln(91 ) and Asn(92) residues is not significant to the stability of the native pr otein. These data collectively demonstrate the central role of Asn(92) in h omodimer interactions. Molecular modelling studies illustrate the favorable packing of the native Asn residue at the dimer interface compared with tha t of the mutant Max peptides.