Structural basis for activation and filamentation of glutaminase

Abstract

Glutaminase catalyzes the first step of glutamine metabolism, converting glutamine to glutamate, which enters the tricarboxylic acid cycle. In human, glutaminase is encoded by two different genes, GLS1 and GLS2 . 1 GLS1 can be alternatively spliced into kidney glutaminase A (KGA) and highly active glutaminase isoform C (GAC) 1 (Fig. 1a ). GAC can be further activated by anions. 2 It has been observed that GAC can form active filaments both in vitro and in vivo. 3 , 4 Glutamine, in addition to glucose, is another vital metabolite for cell. 5 , 6 Glutaminase, especially the GAC isoform, is highly overexpressed in various cancers for the utilization of glutamine. 7 , 8 Currently, a specific glutaminase inhibitor is undergoing several clinical trials. 9 , 10 Activating glutaminase can effectively eliminate cancer cells and solid tumors, and has significant therapeutic potentials. 11 Fig. 1: Functional and structural analysis of glutaminase filament. a Domain organization of KGA and GAC isoforms of human GLS1 gene. KGA and GAC have a common N-terminal signal peptide and a catalytic core (CC) region. After mitochondrial transport, both forms lose the 72 amino acids in the head, leaving a low-complexity sequence from positions 72 to 123. The C-terminal domain of KGA consists of two ankyrin domains, while GAC has a unique C-tail without ankyrin domains. b Glutaminase assay for GAC Apo and GAC Pi with control, demonstrating the activation of glutaminase by Pi. c Negative-stain EM micrographs of GAC Pi incubated at 37 °C for 10 min, showing the filamentation of glutaminase. Scale bar, 100 nm. d Quantitative analysis of negative staining images for GAC Apo and GAC Pi , demonstrating the stimulation of glutaminase filamentation by Pi. e GAC-Pi filaments assembled from GAC tetramers. Rise, twist and GAC tetramer extent are indicated. The helical axis is represented by an orange dash arrow. One helical turn of the filament is displayed, consisting of ~7 helical units with each of them labeled. Left, filament colored by protomers; right, filament highlights one tetramer colored by protomers. Inside a tetramer, the protomers on the same side of the helical axis form a dimer. The interface of two dimers inside a tetramer is referred to as the “dimer–dimer interface” and is indicated by a magenta box. The helical interface and the Pi binding site are highlighted with blue and orange boxes, respectively. The catalytic core in the center of GAC filament and the N-termini on each side are annotated. f Top view of the GAC-Pi filament, demonstrating the helical twist in this orientation. g Zoom-in view of the blue box in ( e ), providing a detailed view of the interactions between GAC tetramers and the formation of the helical filament. Electrostatic interactions are indicated by the dash lines. h Zoom-in view of the magenta box in ( e ), demonstrating the interactions of Pi and L321 from AL with helix of 386–397. The AL, dimer–dimer interface and protomers are colored differently. i Zoom-in view of the red box in ( e ) for the binding site of Pi on the dimer–dimer interface, revealing the composition of the Pi binding pocket. j Glutaminase assay for Q416A Apo and Q416A Pi with GAC Apo and GAC Pi as control. k Comparison of the GAC-PF tetramer and GAC-Pi tetramer models reveals significant conformational changes. l The Y466 loop undergoes a conformational change in the GAC-PF tetramer upon the interaction of the reshaped AL. m Overall conformational changes of the GAC tetramer induced by BPTES or CB-839 binding (PDB IDs: 3UO9 and 5HL1). n Detailed conformational changes of AL upon binding of BPTES or CB-839 in blue and pink, respectively, highlighting the interaction between the inhibitors and AL. Pi is colored by red. The antagonistic interaction mode of Pi and inhibitors with AL is shown. o Agonist Pi promotes the formation of highly active GAC filaments, while the antagonists BPTES and CB-839 stimulate the formation of inactive GAC tetramers. In the highly active filament, the helical interface is located on the catalytic core of GAC, and the tetramers are compressed. When binding antagonists, GAC tetramers present an inhibited expansion mode. p Comparison of binding pockets from representative models. In the GAC-Pi tetramer (PDB ID: 3SS4), the AL is disordered, and the catalytic pocket is wide open. When GAC-Pi forms a filament (PDB ID: 8IMA), the AL is stabilized and the pocket is remodeled, leading to highly active GAC. When the antagonist CB-839 binds to GAC (PDB ID: 5HL1), the AL is fixed away from the pocket, leading to a wide-open pocket and GAC inhibition. AL, dimer interface, dimer–dimer interface, Pi and CB-839 are colored as follows: green, cyan, orange, red and yellow, respectively. Full size image

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