title: Metal-dependent self-assembly of protein tubes from Escherichia coli glutamine synthetase. Cu(2+) EPR studies of the ligation and stoichiometry of intermolecular metal binding sites.
abstract: Escherichia coli glutamine synthetase (GS) is a dodecameric assembly of identical subunits arranged as two back-to-back hexagonal rings. In the presence of divalent metal ions, the dodecamers "stack" along their six-fold axis of symmetry to yield elongated tubes. This self-assembly process provides a useful model for probing metal-dependent protein-protein interactions. However, no direct spectroscopic or structural data have confirmed the identity of the ligands to the shared metal ions in "stacked" GS. Here, 9-GHz Cu(2+) EPR studies have been used to probe the ligand structure and stoichiometry of the metal binding sites. The wild type protein, with N-terminal sequence (His-4)-X(3)-(Met-8)-X(3)-(His-12), exhibits a classic Cu(2+)-nitrogen spectrum, with g = 2.06 G, g = 2.24 G, and A = 19.3 x 10(-3) cm(-1). No superhyperfine structure is observed. The H4C mutant affords a spectrum that is the combination of two spectra at all stages of saturation. One of the overlapping spectra is nearly identical to the spectrum of wild type, and is due to His ligation. The second spectrum observed yields g = 2.28 and A = 17.1 x 10(-3) cm(-1). The linewidth and tensor values of the second component have been assigned to Cu(2+)-S ligation. In contrast, the H12C mutant exhibits an EPR spectrum at low Cu(2+) occupancy that is very similar to the second set of spectral features observed for H4C, and which is assigned to Cu(2+)-S ligation. No Cu(2+)-His ligation is apparent until the Cu(2+)/N-terminal helices ratio is >1.0. At saturation, the g = 2.00-2.06 region of the spectrum is essentially a mirror image of the spectrum obtained with H4C, and is due to overlapping Cu(2+)-N and Cu(2+)-S EPR spectra. The M8L and M8C mutants were also studied, in order to probe the role of position 8 in the N-terminal helix. Spectral parameters of these mutants are nearly identical to each other and to the wild type spectrum at saturating Cu(2+), suggesting that Met-8 does not act as a direct metal ligand. Together, the results provide the first direct evidence for a binuclear metal ion site between each N-terminal helix pair at the GS-GS interface, with both His-4 and His-12 providing metal ligands.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610, USA.
authors.forename: P
authors.initials: P
authors.lastname: Schurke
authors.forename: J C
authors.initials: JC
authors.lastname: Freeman
authors.forename: M J
authors.initials: MJ
authors.lastname: Dabrowski
authors.forename: W M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003300:Copper; D005974:Glutamate-Ammonia Ligase