Structure of the dimeric form of CTP synthase from<i>Sulfolobus solfataricus</i>

Lauritsen, Iben; Willemoës, Martin; Jensen, Kaj Frank; Johansson, E.; Harris, Pernille

doi:10.1107/s1744309110052334

Cited by 24 publications

(23 citation statements)

References 45 publications

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“…The crystal structure of E.coli CTP synthase protein (hereafter called pyrG) revealed that the amino acid residues 15–20 constitute a P-loop-like structure that binds the adenine nucleotide – this provides a structural basis for ATP-dependent tetramerisation of the enzyme [40]. Structural analyses of the human CTPS1 and Sulfolobus solfataricus CTP synthase revealed a similar feature involving the corresponding amino acid residues [41], [42]. Furthermore, CTPsyn amino acids 1–20 could be targets of multiple post-translation modifications.…”

Section: Resultsmentioning

confidence: 99%

Critical roles of CTP synthase N-terminal in cytoophidium assembly

Huang

Wang

Ghosh

et al. 2017

Experimental Cell Research

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Several metabolic enzymes assemble into distinct intracellular structures in prokaryotes and eukaryotes suggesting an important functional role in cell physiology. The CTP-generating enzyme CTP synthase forms long filamentous structures termed cytoophidia in bacteria, yeast, fruit flies and human cells independent of its catalytic activity. However, the amino acid determinants for protein-protein interaction necessary for polymerisation remained unknown. In this study, we systematically analysed the role of the conserved N-terminal of Drosophila CTP synthase in cytoophidium assembly. Our mutational analyses identified three key amino acid residues within this region that play an instructive role in organisation of CTP synthase into a filamentous structure. Co-transfection assays demonstrated formation of heteromeric CTP synthase filaments which is disrupted by protein carrying a mutated N-terminal alanine residue thus revealing a dominant-negative activity. Interestingly, the dominant-negative activity is supressed by the CTP synthase inhibitor DON. Furthermore, we found that the amino acids at the corresponding position in the human protein exhibit similar properties suggesting conservation of their function through evolution. Our data suggest that cytoophidium assembly is a multi-step process involving N-terminal-dependent sequential interactions between correctly folded structural units and provide insights into the assembly of these enigmatic structures.

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Section: Resultsmentioning

confidence: 99%

Critical roles of CTP synthase N-terminal in cytoophidium assembly

Huang

Wang

Ghosh

et al. 2017

Experimental Cell Research

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“…Of course, when the concentration of UTP is below that required for saturation, the dimer–tetamer equilibrium will not lie fully towards the tetramer . As a caveat, it is important to note that the binding of CTP analogues might be sensitive to enzyme concentration, due to dimer–tetramer equilibria and polymerization, although gel filtration‐HPLC revealed that neither the E149D nor the E149Q substitutions adversely affected the ability of these variants to form tetramers at saturating concentrations of UTP (Figure S9). Wild‐type CTPS exhibited three‐times tighter binding with the analogue containing the 2′‐F‐ arabino group (i.e., F‐araCTP) than with that containing the 2′‐F‐ ribo group (i.e., F‐dCTP).…”

Section: Resultsmentioning

confidence: 99%

“…GTP is a positive allosteric effector for Gln‐dependent CTP formation, stimulating both Gln hydrolysis and the subsequent translocation of ammonia to the synthase domain, and functions as a co‐activator with the 4‐phosphorylated UTP intermediate formed during catalysis . The product, CTP, acts as a feedback inhibitor, and promotes both tetramerization of the enzyme and its polymerization into filaments . Because of the variety of nucleotides bound by CTPS, nucleotide analogues have been developed as inhibitors of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…[24] The product, CTP,a cts as af eedback inhibitor, [18] and promotes both tetramerization of the enzyme [25] and itsp olymerization into filaments. [26,27] Because of the varietyo fn ucleotides bound by CTPS, nucleotide analogues have been developeda sinhibitors of the enzyme. Of these inhibitors, [28][29][30] 3-deazauridine-5'-triphosphate (IC 50 % 18 mm) [31] and cyclopentenyl cytosine (CPEC)-5'-triphosphate (IC 50 % 6 mm) [6] are the most extensively studied.…”

Section: Introductionmentioning

confidence: 99%

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Exploring the Potent Inhibition of CTP Synthase by Gemcitabine‐5′‐Triphosphate

et al. 2016

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CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [K =(3.0±0.1) μm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.

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“…Tests of protein production in E. coli BL21(DE3) at 15 °C were also conducted, but yields were generally poorer than in MC1061 at 37 °C, and none of the designs stood out as particularly favored by this strain and temperature. Cultures containing 5 mL or 50 mL of a phosphate-buffered salt medium with the addition of tryptone and yeast extract [60] supplemented with kanamycin (30 μg/mL) were grown to mid-log phase and induced with 1 mM IPTG for 3.5 h at 37 °C or overnight at 15 °C. For large-scale expression, 1-l cultures were grown to late log phase and induced with 1 mM IPTG.…”

Section: Methodsmentioning

confidence: 99%

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

Johansson

Johansen

Christensen

et al. 2016

Journal of Molecular Biology

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Despite the development of powerful computational tools, the full-sequence design of proteins still remains a challenging task. To investigate the limits and capabilities of computational tools, we conducted a study of the ability of the program Rosetta to predict sequences that recreate the authentic fold of thioredoxin. Focusing on the influence of conformational details in the template structures, we based our study on 8 experimentally determined template structures and generated 120 designs from each. For experimental evaluation, we chose six sequences from each of the eight templates by objective criteria. The 48 selected sequences were evaluated based on their progressive ability to (1) produce soluble protein in Escherichia coli and (2) yield stable monomeric protein, and (3) on the ability of the stable, soluble proteins to adopt the target fold. Of the 48 designs, we were able to synthesize 32, 20 of which resulted in soluble protein. Of these, only two were sufficiently stable to be purified. An X-ray crystal structure was solved for one of the designs, revealing a close resemblance to the target structure. We found a significant difference among the eight template structures to realize the above three criteria despite their high structural similarity. Thus, in order to improve the success rate of computational full-sequence design methods, we recommend that multiple template structures are used. Furthermore, this study shows that special care should be taken when optimizing the geometry of a structure prior to computational design when using a method that is based on rigid conformations.

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Structure of the dimeric form of CTP synthase fromSulfolobus solfataricus

Cited by 24 publications

References 45 publications

Critical roles of CTP synthase N-terminal in cytoophidium assembly

Critical roles of CTP synthase N-terminal in cytoophidium assembly

Exploring the Potent Inhibition of CTP Synthase by Gemcitabine‐5′‐Triphosphate

Computational Redesign of Thioredoxin Is Hypersensitive toward Minor Conformational Changes in the Backbone Template

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