title: Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease.
abstract: The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn(2+)-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn(2+) binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein.
authors.affiliation: Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, 6720, Szeged, Hungary.
authors.forename: Eszter
authors.initials: E
authors.lastname: Németh
authors.forename: Tamás
authors.initials: T
authors.lastname: Körtvélyesi
authors.forename: Milan
authors.initials: M
authors.lastname: Kožíšek
authors.forename: Peter W
authors.initials: PW
authors.lastname: Thulstrup
authors.forename: Hans E M
authors.initials: HE
authors.lastname: Christensen
authors.forename: Masamitsu N
authors.initials: MN
authors.lastname: Asaka
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Nagata
authors.forename: Béla
authors.initials: B
authors.lastname: Gyurcsik
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003087:Colicins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D015032:Zinc / Q000378:metabolism
chemical_list: C508089:ColE7 protein, E coli; D003087:Colicins; D050505:Mutant Proteins; D004247:DNA; D015032:Zinc; D000409:Alanine