Our work on cryoEM studies of glutamine synthetase has been recently accepted by Protein Science on March 17th, 2022. This work mainly utilized the powerful cryoEM technique to understand how a dodecameric glutamine synthetase (GS) stacks to others and grows along one dimension to form a long filament. Before the work is online published, here is a brief summary of our work.

We had found that metal ions including nickel ion, zinc ion and cobalt ion can significantly promote the filamentous assembly, however, magnesium, calcium and manganese are unable to change the GS oligomeric states. We applied the advanced cryoEM data process including helical reconstruction and focus refinement using UI-friendly cryoSPARC 3.2 to conquer the 50-year unresolved mechanism. The nickel-induced GS filament has many stacked GS through a face-to-face contact. Below has 2 images of cryoEM maps. The left one is the map determined by helical reconstruction approach using cryoSPARC 3.2. The right one is the local resolution distribution of GS filament determined by single particle reconstruction in cryoSPARC 3.2. Maps are identical but the SPA-map is sharper (2.94 Å) and the central GS 12mer is of high quality.

The interface is actually simple — the first 13 residues of GS are in charge of the stacking and assembly. The first 13 residues of GS form an alpha-helix which we called helix 1, H1. Six H1 from one dodecameric GS (GS-A) contact to another 6 helix H1 from GS-B in a parallel alignment whereas a nickel ion coordinates His5, Met9, Glu12 sidechain atoms to tightly bring two helices H1 closely. Moreover His13 from GS-A and Glu12 from GS-B form a hydrogen bond meaning two hydrogen bonds at each hinged H1-H1 interface. As one “face” of GS is composed of six helices H1, there are 6 nickel ions bridging two hexagonal ring-like GS faces together. We calculated the rotation angle as of 18 degrees between two stacked dodecameric GS proteins. The cryoEM structure of stacked dodecameric GS is available at RCSB PDB site (accession ID: 7W85). By repeating GS stacking 20 times, the filamentation accomplishes a full rotation yielding a ~200 nm crossover distance.

We then mutated residues His5 and His13 separately or simultaneously to Ala to confirm the assembly. Both negative stained EM and dynamic light scattering (DLS) data present that the two histidine residues His5 and His13 are crucial for forming GS filament. Double mutation of H5A-H13A completely diminishes the capability of filament formation.

In short, we determined the GS filament structure and unraveled the mechanism since the first report in 1968 by E.R. Stadtman (Biochemistry 1968). The work provides information related to stress-induced conditions and could explain low metabolic/biosynthetic activities of bacteria when meeting harsh conditions.