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Metal-dependent Self-assembly of Protein Tubes from Escherichia coli Glutamine Synthetase

Peter Schurke,J. Freeman,M. Dabrowski,W. Atkins

Published 1999 in Journal of Biological Chemistry

ABSTRACT

Escherichia coliglutamine 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 Cu2+ 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 Cu2+-nitrogen spectrum, with g⊥ = 2.06 G, g∥ = 2.24 G, and A∥= 19.3 × 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 × 10−3 cm−1. The linewidth and tensor values of the second component have been assigned to Cu2+-S ligation. In contrast, the H12C mutant exhibits an EPR 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 ligation. 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 essentially a mirror image of the spectrum obtained with H4C, and is due to overlapping Cu2+-N and Cu2+-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 Cu2+, 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.

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