Three-dimensional Structures Of Biological Macromolecule

Heart, Lung, And Blood Institute

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Abstract

The three-dimensional structure of the wild type and mutants of homeodomains in the NK-2 class, specifically the parent NK-2 homeodomain, and the full length NKX-2.5, NKx-2.8 and NKX-3.1 homeodomain containing proteins, both in the free state and bound to an uncommon 18 base-pair DNA consensus segment are being investigated. In addition, studies of the protein-protein complex made up of the NKX-2.5 and GATA-4 proteins have been initiated. Studies on mutant NKX-2.5 homeodomains associated with atrial septum defect show that either the tertiary structures of the mutant proteins are altered or their binding to the functional DNA binding sites are altered. For example, replacement of an invariant asparagine in position 51 of the homeodomain by lysine reduces the binding affinity by a factor of 1000 and alters the specificity of the protein-DNA interaction. Preliminary results suggest that this mutant results in a significant alteration of the preferred target DNA. Structural studies demonstrate that the modified homeodomain maintains the characteristic three-dimensional structure seen for most homeodomains. Thermodynamic measurements on the same wild type and mutant homeodomains are in progress. The three-dimensional structure of the NK-2 homeodomain bound to DNA, where the tyrosine in position 54 is replaced by a methionine, shows that the methionine with its longer side-chain also alters the specificity of the homeodomain-DNA interaction. The structure of another modified NK-2 homeodomain bound to DNA, where alanine in position 35 is replaced by threonine, results in a significantly altered protein structure. This structural alteration constitutes the molecular basis of early embryonic lethality associated with the mutation in the corresponding gene. Transgenic studies on the tyrosine to methionine modified homeodomain are nearing completion.