January 25, 2024: Prof Eric Greene, San Francisco State University

Decoding the Complexity of Human Glutamine Synthetase: Insights from Cryo-EM and Ensemble Analysis
Here, we delve into the regulatory mechanisms and associated structural alterations of human glutamine synthetase (GS). GS is a key enzyme in nitrogen metabolism and glutamine biosynthesis and is essential for ammonia detoxification and glutamate level regulation in the brain. We used cryo-electron microscopy (cryo- EM), molecular dynamics simulations, and 3D variability analysis (3DVA) to create structural ensembles of GS 'in action', unveiling a dynamic compositional and conformational landscape with implications for allosteric regulation, catalytic activity, and disease variants.
We found that GS forms head-on-head filament structures in the apo state that can be further stabilized by glutamine binding directly at the interface. High-resolution cryo-EM structures (1.97 - 2.3 Å) showed variable density corresponding to the active site loop in GS, demonstrating its inherent flexibility. Molecular dynamics ensemble analysis revealed a large conformational space for the active site loop, which is comparatively restrained in non-filamentous cryo-EM maps. Steady-state kinetics demonstrate that filamentation directly affects the KM for ammonia, implicating filamentation as a rapid feedback regulation mechanism for GS.
We further applied 3DVA to create cryo-EM map ensembles of non-filamentous GS under turnover conditions. The observed conformational differences between filamentous and non-filamentous compositions could be replicated in the conformational landscape of solely non-filamentous composition, showing that filamentation results in a biased conformational ensemble rather than static conformational switching. We refined 3DVA generated particle clusters individually (2.1 - 2.6 Å resolution range) and identified correlated amino acids whose conformational changes may underlie allosteric stabilization of the active site loop. Mutagenesis of these correlative amino acids led to decoupling of the GS two-step reaction scheme, implicating their role in an allosteric network. Cryo-EM reconstructions of mutant GS showed widespread structural perturbations resulting from disruption of this allosteric network.
Finally, we incorporated human GS experimentation into a Course-based Undergraduate Research Experience (CURE), where students purify and perform initial characterization of GS disease variants and generate hypotheses before their enzyme preparations are subjected to cryo-EM structural characterization.
In summary, our research provides a revised understanding of the regulatory mechanisms and associated structural changes of human GS during catalysis, and further, demonstrates the utility of cryo-EM towards ensemble-function analysis of enzyme complexes.