Supramolecular Self-Assembly of Glutamine Synthetase: Mutagenesis of a Novel Intermolecular Metal Binding Site Required for Dodecamer Stacking

Abstract

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 approximately 3.5 A 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…We have suggested previously, based on turbidity measurements, that Cu 2ϩ induces GS dodecamers to self-assemble into extended tubes (8,13). The self-assembly process was monitored by light scattering and transmission EM.…”

“…Mutagenesis and Protein Purification-Site-directed mutagenesis and protein purification were as described previously (13), with the following modifications: ZnSO 4 precipitation, acetone precipitation, and (NH 4 ) 2 SO 4 /acid precipitation steps were omitted. Instead, after treatment with streptomycin sulfate, the cellular lysate was chromatographed over Blue Sepharose CL-6B (Amersham Pharmacia Biotech), and eluted with 40 ml of 20 mM ADP.…”

“…In addition, we have explored in vitro methods for removing the biologically imposed symmetry of the GS dodecamer, which allow for control over the length of GS tubes (12). Also, the solution conditions that influence this metal-dependent protein-protein interaction have been previously characterized for wild type and site-directed mutants of E. coli GS, in order to understand factors that control this process (8,13), and the thermodynamics of the reaction have been partially described (14). Together, these results have yielded GS variants with novel self-assembly properties and a useful structural model for the molecular mechanism of recognition between GS dodecamers.…”

“…In addition, the Met-8 side chains may provide ligands to either or both metals. This is based on the observation that the M8L mutant stacks poorly with all metal ions, whereas the M8C mutant stacks efficiently with some metals and poorly with others (13).…”

“…Previously, it was suggested that Met-8 may play a role in the stabilization of these two sites (13). In an effort to elucidate the contribution from this residue, the M8C and M8L mutants were studied by EPR.…”