Metal-dependent self-assembly of protein tubes from Escherichia coli glutamine synthetase - Cu2+ EPR studies of the ligation and stoichiometry of intermolecular metal binding sites

Escherichia coli glutamine synthetase (GS) is a dodecameric assembly of ide ntical subunits arranged as two back-to-back hexagonal rings. In the presen ce 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. Ho wever, no direct spectroscopic or structural data have confirmed the identi ty of the ligands to the shared metal ions in "stacked" GS, Here, 9-GHz Cu2 + EPR studies have been used to probe the ligand structure and stoichiometr y of the metal binding sites. The wild type protein, with N-terminal sequen ce (His-4)-X-3-(Met-8) -X-3-(His-12), exhibits a classic Cu2+-nitrogen spec trum, with g(perpendicular to) = 2.06 G, g(parallel to) = 2.24 G, and A(par allel to) = 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 identic al to the spectrum of wild type, and is due to His ligation, The second spe ctrum observed yields g(parallel to) = 2.28 and A(parallel to) = 17.1 x 10( -3) cm(-1). The linewidth and tensor values of the second component have be en assigned to Cu2+-S ligation. In contrast, the H12C mutant exhibits an EP R spectrum at low Cu2+ occupancy that is very similar to the second set of spectral features observed for H4C, and which is assigned to Cu2+-S ligatio n, No Cu2+-His ligation is apparent until the Cu2+/N-terminal helices ratio is > 1.0, At saturation, the g = 2.00-2.06 region of the spectrum is essen tially a mirror image of the spectrum obtained with H4C, and is due to over lapping Cu2+-N and Cu2+-S EPR spectra, The M8L and M8C mutants were also st udied, in order to probe the role of position 8 in the N-terminal helix. Sp ectral parameters of these mutants are nearly identical to each other and t o the wild type spectrum at saturating Cu2+, suggesting that Met-8 does not act as a direct metal ligand, Together, the results provide the first dire ct evidence for a binuclear metal ion site between each N-terminal helix pa ir at the GS GS interface, with both His-4 and His-12 providing metal ligan ds.