title: Control of enzyme reaction by a designed metal-ion-dependent α-helical coiled-coil protein.
abstract: Regulation of protein function by external stimuli is a fascinating target for de novo design. We have constructed a peptide that assembles into a homotrimer in the presence of metal ions, such as Ni(2+), Cu(2+), and Zn(2+). We fused the peptide construct to the DNA-binding domain (DBD) of the heat shock factor from Saccharomyces cerevisiae, which binds tandem repeats of the heat shock element (HSE). However, the fusion protein bound to the natural three tandem HSEs even in the absence of metal ions, although mainly as the dimerized protein. Using "skipped" HSEs containing six additional nucleotides inserted between two adjacent HSEs, to prevent interactions between the DBDs, we found the fusion protein bound to the new DNA target in a metal-ion-dependent manner, as monitored by a HindIII protection assay. The fusion protein containing two metal binding sites in the metal-ion-controlled domain inhibited RNA transcription by T7 RNA polymerase in the presence of metal ions, in a template containing skipped HSEs downstream of the T7 promoter. The designed protein therefore regulates the functions of the enzyme in a metal-ion-dependent manner.
authors.affiliation: Department of Material Sciences, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-chou, Nagoya, 466-8555, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Murase
authors.forename: Sonoko
authors.initials: S
authors.lastname: Ishino
authors.forename: Yoshizumi
authors.initials: Y
authors.lastname: Ishino
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D008670:Metals; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA