No abstract

Fibroin-modulator-binding protein 1 (FMBP-1) is a factor that binds the transcriptional activation elements of the fibroin gene. It has a novel structure, consisting of four tandem repeats (R1-R4) of 23 amino acids each in the C-terminal half. This region is referred to as the STPR (score and three amino acid peptide repeat) domain and acts as a DNA-binding domain in FMBP-1. Interestingly, the homology among the four repeats is remarkably high. Here, we have determined the three-dimensional structures of the four repeats by NMR. All four repeat units have basically the same structure: a short alpha-helix in the N-terminal half maintained by a salt bridge and an N-capping box. CD studies showed that the full-length STPR domain was 31% helical in solution. This is explained by the connections among the four short helices that were determined separately by NMR. From the thermal-denaturation study, it can be deduced that these four helices in the full-length STPR domain moved flexibly with no interaction among them. However, the specific DNA caused a distinct increase, of up to 76%, in the alpha-helical content of the full-length STPR domain. This finding suggests that the binding of the full-length STPR domain to specific DNA causes an induced-fit conformational change that increases alpha-helicity; the poorly structured regions of the protein may form a regular secondary structure. Furthermore, the mutation analysis showed that the four repeats of the STPR domain raise the possibility of interaction with DNA in different ways.

Fibroin-modulator-binding protein 1 (FMBP-1) is a predicted transcription factor of the silkworm fibroin gene. The DNA-binding domain of FMBP-1 consists of four almost perfect tandem repeats of 23 amino acids each (R1-R4), and is referred to as the score and three amino acid peptide repeat (STPR) domain. This characteristic domain is conserved in eukaryotes, but the DNA-binding mode is not known. In this study, the structural properties of the DNA-bound form of the STPR domain were characterized. The combined experiments indicated that the STPR domain bound to the DNA duplex with a 1:1 binding ratio. The specific DNA caused considerable changes in the thermal unfolding profile and the digestion pattern of the STPR domain. These data suggested that the domain adapts a quite rigid helix-rich structure in the DNA-bound state, even though it moves flexibly in the absence of DNA. Furthermore, mutual induced-fit conformational change was also observed in DNA. Finally, we determined the DNA-binding surface of the STPR third repeat (R3) by alanine scanning mutagenesis; a particular site, composed of hydrophobic and hydrophilic residues, was identified. Notably, the substitution of Arg-9 in R3 with alanine residue, which is located in the middle of the surface, drastically abolished the alpha-helix-inducing and DNA-binding abilities. From these results, we predicted the DNA-binding mode of the STPR domain.

The antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, were successfully detected in the aorta of spontaneously hypertensive rats after orally administering these peptides by a guanidine-thiocyanate treatment to prevent proteolysis. Cy3-labeled versions of both peptides were localized in the endothelial cells of arterial vessels in the rats. The accumulation of both peptides in the endothelial cells suggested in vivo inhibitory activity of the angiotensin I-converting enzyme.