“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

McCluskey, Gregory D.; Bearne, Stephen L.

doi:10.1016/j.bbagen.2018.08.008

Cited by 10 publications

(9 citation statements)

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“…Indeed, point mutations that impair GTP binding have been shown to lead to a decrease in activity and uncoupling between the NH 3 derived from glutamine hydrolysis and CTP formations (12,30,31). Moreover, it has been demonstrated that GTP inhibits NH 3 -dependent CTP synthesis in a concentrationdependent manner in the absence of glutamine, supporting the hypothesis that exogenous NH 3 may enter the tunnel through the GTP binding site (13,32).…”

Section: Gtp Binding Mode Suggests Its Role In Preventing Leakage Of Nascentmentioning

confidence: 89%

“…On the other hand, His55 (His57 of ecCTPS) was predicted to act as a gate at the exit of the ammonia tunnel (10,13,17,26). It was suggested that when CTPS is bound with substrates, the interaction between UTP and His55 alters the orientation of His55, causing the gate to open.…”

Section: Conformational Switch Of Ammonia Tunnel Offers Insight Intomentioning

confidence: 99%

“…A few mechanisms are proposed from structural and biochemical studies to control the pace of the reaction, as follows. First, GTP appears to promote channeling of NH 3 derived from glutamine hydrolysis to the synthase site by preventing the ammonia tunnel from being constricted or leaky (11)(12)(13). Second, the "gate" residue of the ammonia tunnel controls the access to the ALase active site, which is available to NH 3 only when bound by UTP (10).…”

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confidence: 99%

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Structural basis for ligand binding modes of CTP synthase

Zhou

Guo

Chang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Cytidine triphosphate synthase (CTPS), which comprises an ammonia ligase domain and a glutamine amidotransferase domain, catalyzes the final step of de novo CTP biosynthesis. The activity of CTPS is regulated by the binding of four nucleotides and glutamine. While glutamine serves as an ammonia donor for the ATP-dependent conversion of UTP to CTP, the fourth nucleotide GTP acts as an allosteric activator. Models have been proposed to explain the mechanisms of action at the active site of the ammonia ligase domain and the conformational changes derived by GTP binding. However, actual GTP/ATP/UTP binding modes and relevant conformational changes have not been revealed fully. Here, we report the discovery of binding modes of four nucleotides and a glutamine analog 6-diazo-5-oxo-L-norleucine in Drosophila CTPS by cryo–electron microscopy with near-atomic resolution. Interactions between GTP and surrounding residues indicate that GTP acts to coordinate reactions at both domains by directly blocking ammonia leakage and stabilizing the ammonia tunnel. Additionally, we observe the ATP-dependent UTP phosphorylation intermediate and determine interacting residues at the ammonia ligase. A noncanonical CTP binding at the ATP binding site suggests another layer of feedback inhibition. Our findings not only delineate the structure of CTPS in the presence of all substrates but also complete our understanding of the underlying mechanisms of the allosteric regulation and CTP synthesis.

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Section: Gtp Binding Mode Suggests Its Role In Preventing Leakage Of Nascentmentioning

confidence: 89%

Section: Conformational Switch Of Ammonia Tunnel Offers Insight Intomentioning

confidence: 99%

mentioning

confidence: 99%

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Structural basis for ligand binding modes of CTP synthase

Zhou

Guo

Chang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…The S-state CTPS2 filament structure is the first near-atomic resolution structure of any CTPS in the substrate-bound conformation, providing insight into the mechanism of ammonia transfer between the two active sites. Previous studies have identified a putative ~25 Å tunnel required to facilitate ammonia transfer between the glutaminase and amidoligase active sites 9,24 (Fig. 3, Extended Data Fig.…”

Section: Substrate Binding Opens a Tunnel In The Ctps Monomermentioning

confidence: 99%

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Lynch

Kollman

2019

Nat Struct Mol Biol

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Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating activity. Recent studies have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments. Here we describe an ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). Cryo-EM structures reveal that CTPS2 filaments dynamically switch between active and inactive forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures reveal a link between conformation and control of ammonia channeling between the enzyme's active sites, and explain differences in regulation of human CTPS isoforms. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.CTP synthase (CTPS) is the key regulatory enzyme in pyrimidine biosynthesis, with critical roles in regulation of nucleotide balance 1 , maintenance of genome integrity 2,3 , and synthesis of membrane phospholipids 4 . CTPS catalyzes the conversion of UTP to CTP in an ATPdependent process, the rate-limiting step in CTP synthesis. CTPS is regulated through feedback inhibition by CTP binding, and is allosterically regulated by GTP, making it sensitive to levels of the four essential ribonucleotides, reflecting its role as a critical regulatory node in nucleotide metabolism [5][6][7][8] . CTPS is a homotetramer, with each monomer composed of a glutaminase and an amidoligase domain connected by a helical linker 9 . Ammonia is generated from glutamine then transfered to the amidoligase domain, where it is ligated to UTP to form CTP; while both of these catalytic mechanisms are well understood, the mechanism of ammonia transfer between the two separated active sites has not yet been described. Previously, we showed that CTPS undergoes a conserved conformation cycle controlled by substrate and product binding, involving two major Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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Section: Resultsmentioning

confidence: 99%

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Lynch

Kollman

2019

Preprint

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Many enzymes assemble into defined oligomers, providing a mechanism for 8 cooperatively regulating enzyme activity. Recent studies in tissues, cells, and in vitro 9 have described a mode of regulation in which enzyme activity is modulated by 10 polymerization into large-scale filaments 1-5 . Enzyme polymerization is often driven by 11 binding to substrates, products, or allosteric regulators, and tunes enzyme activity by 12 locking the enzyme in high or low activity states 1-5 . Here, we describe a unique, 13 ultrasensitive form of polymerization-based regulation employed by human CTP 14 synthase 2 (CTPS2). High-resolution cryoEM structures of active and inhibited CTPS2 15 filaments reveal the molecular basis of this regulation. Rather than selectively stabilizing 16 a single conformational state, CTPS2 filaments dynamically switch between active and 17 inactive filament forms in response to changes in substrate and product levels. Linking 18 the conformational state of many CTPS2 subunits in a filament results in highly 19 cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 20 tetramer alone. The structures also reveal a link between conformational state and 21 control of ammonia channeling between the enzyme's two active sites. This filament-22 based mechanism of enhanced cooperativity demonstrates how the widespread 23 phenomenon of enzyme polymerization can be adapted to achieve different regulatory 24 outcomes. 25 26 CTP synthase (CTPS) is the key regulatory enzyme in pyrimidine biosynthesis, with critical roles 27 in regulation of nucleotide balance 6 , maintenance of genome integrity 7,8 , and synthesis of 28 membrane phospholipids 9 . CTPS catalyzes the conversion of UTP to CTP in an ATP-dependent 29 process, the rate-limiting step in CTP synthesis. CTPS is regulated through feedback inhibition 30 by CTP binding, and is allosterically regulated by GTP, making it sensitive to levels of the four 31 essential ribonucleotides, reflecting its role as a critical regulatory node in nucleotide 32 metabolism 10-13 . CTPS is a homotetramer, with each monomer composed of a glutaminase and 33 an amidoligase domain connected by a helical linker 14 . Ammonia is generated from glutamine 34 then transfered to the amidoligase domain, where it is ligated to UTP to form CTP; while both of 35 these catalytic mechanisms are well understood, the mechanism of ammonia transfer between 36 the two separated active sites has not yet been described. Previously, we showed that CTPS 37 undergoes a conserved conformation cycle controlled by substrate and product binding, 38 involving two major structural changes: upon substrate binding, the glutaminase domain rotates 39 towards the amidoligase domain, bringing the two active sites closer, and the tetramer interface 40 rearranges to accommodate UTP binding 5 . 41Humans have two CTPS isoforms encoded on separate genes, CTPS1 and CTPS2, that 42 share 75% identity. Their relative roles remain unclear. CTPS1 plays a specific and central role 43 in lymphocyte ...

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“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Cited by 10 publications

References 91 publications

Structural basis for ligand binding modes of CTP synthase

Structural basis for ligand binding modes of CTP synthase

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

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