Kinetic analysis of Drosophila Vnd protein containing homeodomain with its target sequence

Abstract

Homeodomain (HD) is a highly conserved DNA-binding domain composed of helix-turn-helix motif. Drosophila Vnd (Ventral nervous system defective) containing HD acts as a regulator to either enhance or suppress gene expression upon binding to its target sequence. In this study, kinetic analysis of Vnd binding to DNA was performed. The result demonstrates that DNA-binding affinity of the recombinant protein containing HD and NK2-specific domain (NK2-SD) was higher than that of the full-length Vnd. To access whether phosphorylation sites within HD and NK2-SD affect the interaction of the protein with the target sequence, alanine substitutions were introduced. The result shows that S631A mutation within NK2-SD does not contribute significantly to the DNA-binding affinity. However, S571A and T600A mutations within HD showed lower affinity for DNA binding. In addition, DNA-binding analysis using embryonic nuclear protein also demonstrates that Vnd interacts with other nuclear proteins, suggesting the existence of Vnd as a complex. [BMB reports 2010; 43(6): 407-412]

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

“…HD proteins recognize a canonical core sequence 5'-TAAT-3', which plays a major role in determining high binding affinity with an approximate K d value of 10 -8 to 10 -11 M, and regulate the expression of a target gene acting either as an activator or a repressor (7-9). In contrast, ABD-B HD binds preferentially to a sequence with an unusual 5'-TTAT-3' core motif instead of 5'-TAAT-3' for other HD proteins (10).…”