SUPRAMOLECULAR SELF-ASSEMBLY OF GLUTAMINE-SYNTHETASE - MUTAGENESIS OFA 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 se veral divalent metal ions. The molecular mechanism for this metal-indu ced self-assembly of the E. coli GS has been studied by molecular mode ling 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 compleme ntary fit, based on steric constraints, reveals a possible interaction between the N-terminal helices from adjacent dodecamers. The amino ac id 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 Ang strom of the analogous side chains in the subunits from the adjacent d odecamer in the stacked complex. His-4, Met-8, and His-12 from adjacen t helices provide potential ligands for a binuclear metal binding site . Replacement of each of these surface residues with aliphatic amino a cids has negligible effects on the enzymatic activity, the regulation of activity via adenylylation, and gross dodecameric structure. Howeve r, the rate and extent of metal ion-mediated self-assembly of GS tubul es are reduced to <2% of the wild-type protein in the single mutants H 4A, H12L, and H12D. The M8L mutant demonstrates a 3-fold decrease in t he bimolecular rate constant for stacking, but electron microscopy ind icates that this mutant does form stacked tubes. The cysteine-containi ng mutants H4C, M8C, and H12C were also constructed. The stacking abil ity is partially recovered in the H12C and H4C mutant proteins, and th e M8C protein stacks with an apparent bimolecular rate constant that i s 8-fold faster and 2-fold faster than that of the wild-type protein w ith Zn2+ and Co2+, respectively. In contrast, the M8C mutant does not stack with Cu2+, The interface between dodecamers within the complex i s dominated by polar residues, and the proposed model indicates that t he interface remains partially solvated. These results indicate that t he mechanism of self-assembly of E. coli GS to form stacked tubules in volves an interdodecameric metal binding site formed from histidine re sidues at the i and i + 8 positions of two adjacent helices and a meth ionine at i + 4.