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 Myc oncoprotein. The pivotal role of Max within the Myc regulatory network is dependent upon its ability to dimerize via the helix-loop-helix leucine zipper domain. The Max homodimer contains a tetrad of polar residues at the interface of the leucine zipper domain. A conserved interfacial Asn residue at an equivalent position in two other leucine zipper proteins has been shown to decrease homodimer stability. The unusual arrangement of this Gln-Asn/Gln'-Asn' tetrad prompted us to investigate whether Asn(92) plays a similar role in destabilizing the Max homodimer. This residue was sequentially replaced with aliphatic and charged residues. Thermal denaturation, redox time course and analytical ultracentrifugation studies show that the N92V mutation does not increase homodimer stability. Replacing this residue with negatively charged side chains in N92D and N92E destabilizes the mutant homodimer. 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 protein. These data collectively demonstrate the central role of Asn(92) in homodimer interactions. Molecular modelling studies illustrate the favorable packing of the native Asn residue at the dimer interface compared with that of the mutant Max peptides.
Tchan
M C
MC
Department of Biochemistry, University of Sydney, Sydney, New South Wales 2006, Australia.
Choy
K J
KJ
Mackay
J P
JP
Lyons
A T
AT
Bains
N P
NP
Weiss
A S
AS
D000577:Amides / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers; D019281:Dimerization; D018257:Helix-Loop-Helix Motifs; D016350:Leucine Zippers / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D010084:Oxidation-Reduction; D011489:Protein Denaturation
D000577:Amides; D017931:DNA Primers; D001216:Asparagine