Abstract 5270: Crystal Structure of Nkx2.5 Homeodomain in Complex with Anf-242 Motif

Abstract

NKX2.5 is a homeodomain (HD) containing transcription factor preferentially expressed in cardiomyocytes. Mutations in the NKX2.5 gene have been identified in patients with congenital heart disease, suggesting its critical roles in cardiac development and maturation. NKX2.5 is reported to regulate transcription of atrial natriuretic factor (ANF), potentially through direct binding to -242 bp proximal promoter region (ANF-242). ANF-242 contains two palidromic NKX2.5 binding sites, and occupation of both sites by NKX2.5 has been reported. In addition, physical interaction between NKX2.5 proteins has been suggested to facilitate DNA binding through domains outside of the HD. However, detailed crystal structure of NKX2.5 protein is currently not available. The HD of human NKX2.5 (aa 138–197; C193S) with the oxidizable Cys193 substituted with Ser was co-crystallized with 19bp double stranded DNA including ANF-242 region. This mutant was reported to have the same DNA binding characteristics as the wild type. The crystal structure was determined at 1.8 Å resolution with final R-working and R-free values of 0.234 and 0.278, respectively. Consistent with previous studies, two molecules of NKX2.5 HD are bound to ANF-242 region without physical interaction between the two proteins. Column purification demonstrated that NKX2.5 behaves as dimers in solution in a concentration dependent manner. These results suggest that dimeric NKX2.5 HD in solution may change conformation upon DNA binding. Among various NK class proteins, the three-dimensional structure has only been determined in the HD of Drosophila vnd/NK2 (ventral nervous system defective proteins) using NMR. When the two HDs are compared, NKX2.5 HD shows extended DNA contacts to a total of 8 base pairs. Most of the residues of HD implicated in pathology of mutants are involved in DNA binding and structural stabilization. The structure of NKX2.5 HD with ANF-242 shows that the HD occupies two binding sites of ANF-242 without physical interaction between themselves. This high resolution crystal structure of NKX2.5 protein provides a detailed picture of protein and DNA interactions, which allows us to predict DNA binding of mutants identified in human patients. This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).