SUPRAMOLECULAR SELF-ASSEMBLY OF GLUTAMINE-SYNTHETASE - MUTAGENESIS OF A NOVEL INTERMOLECULAR METAL-BINDING SITE REQUIRED FOR DODECAMER STACKING

Dodecameric glutamine synthetase (GS) from Escherichia coli assembles into highly ordered supramolecular protein tubes in the presence of several divalent metal ions. The molecular mechanism for this metal-induced self-assembly of the E. coli GS has been studied by molecular modeling and site-directed mutagenesis. The X-ray crystal structure of the nearly identical Salmonella typhimurium GS has been used to construct a model of the ''stacked'' complex between two dodecamers. A complementary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino acid side chains of His and Met residues within the helices from each of the subunits of one face of a dodecamer lie within similar to 3.5 Angstrom of the analogous side chains in the subunits from the adjacent dodecamer in the stacked complex. His-4, Met-8, and His-12 from adjacent helices provide potential ligands for a binuclear metal binding site. Replacement of each of these surface residues with aliphatic amino acids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. However, the rate and extent of metal ion-mediated self-assembly of GS tubules are reduced to <2% of the wild-type protein in the single mutants H4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in the bimolecular rate constant for stacking, but electron microscopy indicates that this mutant does form stacked tubes. The cysteine-containing mutants H4C, M8C, and H12C were also constructed. The stacking ability is partially recovered in the H12C and H4C mutant proteins, and the M8C protein stacks with an apparent bimolecular rate constant that is 8-fold faster and 2-fold faster than that of the wild-type protein with Zn2+ and Co2+, respectively. In contrast, the M8C mutant does not stack with Cu2+, The interface between dodecamers within the complex is dominated by polar residues, and the proposed model indicates that the interface remains partially solvated. These results indicate that the mechanism of self-assembly of E. coli GS to form stacked tubules involves an interdodecameric metal binding site formed from histidine residues at the i and i + 8 positions of two adjacent helices and a methionine at i + 4.