Effect of arginine mutation of alanine-556 on DNA recognition of zinc finger protein Sp1 Original Research Article

Human transcription factor Sp1, which contains three Cys2His2-class zinc finger motives, specifically binds to the so-called GC box DNA. It has been indicated that finger 1 has a unique DNA-binding mode compared with fingers 2 and 3, or the Zif268 model. Therefore, we investigate the role of Ala at position 6 on the recognition helix, which is not responsible for guanine recognition and highly conserved among Sp1 family. Several Ala-556 mutations of Sp1 bind to DNA with different DNA-binding features. In particular, the Ala→Arg substitution alters the DNA-binding contribution of the three zinc fingers in Sp1. In this case, the DNA-binding specificity of each finger decreases in the order 2>1>3. This result reveals that one amino acid in position 6 plays an important role not only for the selectivity to the putative finger 1 subsite, but also for the binding mode of the three fingers to each finger subsite. Probably, Ala-556 is indispensable to characterize the binding mode of the Sp1 zinc fingers, namely the diverse binding contribution of finger 1 and the rigid binding one of finger 3. In Sp1, the N-terminal finger 1 serves as a ‘hinge finger’.