Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism

Abstract

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGA's active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.Warburg effect | RAS/MAPK | crystallography T he Warburg effect in cancer biology describes the tendency of cancer cells to take up more glucose than most normal cells, despite the availability of oxygen (1, 2). In addition to altered glucose metabolism, glutaminolysis (catabolism of glutamine to ATP and lactate) is another hallmark of cancer cells (2, 3). In glutaminolysis, mitochondrial glutaminase catalyzes the conversion of glutamine to glutamate (4), which is further catabolized in the Krebs cycle for the production of ATP, nucleotides, certain amino acids, lipids, and glutathione (2, 5).Humans express two glutaminase isoforms: KGA (kidney-type) and LGA (liver-type) from two closely related genes (6). Although KGA is important for promoting growth, nothing is known about the precise mechanism of its activation or inhibition and how its functions are regulated under physiological or pathophysiological conditions. Inhibition of rat KGA activity by antisense mRNA results in decreased growth and tumorigenicity of Ehrlich ascites tumor cells (7), reduced level of glutathione, and induced apoptosis (8), whereas Myc, an oncogenic transcription factor, stimulates KGA expression and glutamine metabolism (5). Interestingly...

Active Glutaminase C Self-assembles into a Supratetrameric Oligomer That Can Be Disrupted by an Allosteric Inhibitor

“…Robinson et al (6) first demonstrated biochemically that BPTES inhibits the GLS1 isoform (but not the liver-type isozyme, LGA/GLS2) by interfering with its phosphate-dependent allosteric activation and promoting the stabilization of the glutaminase into an inactive tetrameric form, later confirmed structurally (7,8). Our collection of results indicates that BPTES inhibits GAC by trapping the gating loop at a rigid open conformation, which in turn prevents superoligomer formation.…”

“…respectively (7,8), our crystal form presents a key unique feature. As a consequence of the binding of the small molecule to the tetramer interface, the main chain of the gating loop-consistently poorly ordered across all the GAC crystallographic models-is in a stable open conformation in all four monomers.…”

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…The structures of both GLS and LGA have been determined by x-ray crystallography, although substantially more effort has been devoted to describing GLS than LGA. Table 3 shows the details of the currently available mammalian glutaminase crystal structures [37,[63][64][65][66][67][68]. The constructs used for crystallography include the isolated glutaminase catalytic domain (e.g., 3CZD or 3VP1) and the entire biologically processed form of the enzyme (e.g., 3UNW or 5FI7), and structures have been determined with glutamine, glutamate or assorted inhibitors bound.…”

“…Figure 6A) [61,124]. BPTES is a long, highly flexible and C 2 -symmetrical molecule which binds in a 1:2 ratio with GLS and sits at the dimer-dimer binding interface, where it stabilizes an inactive tetramer ( Figure 6B) [67,124]. It has been suggested to have approximately 1000-fold greater affinity for GLS than for LGA, however to date, the only studies showing this with purified recombinant enzymes have used mutants of GLS which resemble LGA at the BPTES binding site, rather than using full-length LGA [68,123].…”

A pathway map of glutamate metabolism

“…cell growth and proliferation (Thangavelu et al 2012). energy metabolism and protection against antioxidants (Hu et al 2010).…”