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Structural basis for the helical filament formation of Escherichia coli glutamine synthetase.

ABSTRACT: Escherichia coli glutamine synthetase (EcGS) spontaneously forms a dodecamer that catalytically converts glutamate to glutamine. EcGS stacks with other dodecamers to create a filament-like polymer visible under transmission electron microscopy. Filamentous EcGS is induced by environmental metal ions. We used cryo-electron microscopy (cryo-EM) to decipher the structure of metal ion (nickel)-induced EcGS helical filament at a sub-3Å resolution. EcGS filament formation involves stacking of native dodecamers by chelating nickel ions to residues His5 and His13 in the first N-terminal helix (H1). His5 and His13 from paired parallel H1 helices provide salt bridges and hydrogen bonds to tightly stack two dodecamers. One subunit of the EcGS filament hosts two nickel ions, whereas the dodecameric interface and the ATP/Mg-binding site both host a nickel ion each. We reveal that upon adding glutamate or ATP for catalytic reactions, nickel-induced EcGS filament reverts to individual dodecamers. Such tunable filament formation is often associated with stress responses. Our results provide detailed structural information on the mechanism underlying reversible and tunable EcGS filament formation.

SUBMITTER: Huang PC

PROVIDER: S-EPMC8996467 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Structural basis for the helical filament formation of Escherichia coli glutamine synthetase.

Huang Pei-Chi PC Chen Shao-Kang SK Chiang Wei-Hung WH Ho Meng-Ru MR Wu Kuen-Phon KP

Protein science : a publication of the Protein Society 20220501 5

Escherichia coli glutamine synthetase (EcGS) spontaneously forms a dodecamer that catalytically converts glutamate to glutamine. EcGS stacks with other dodecamers to create a filament-like polymer visible under transmission electron microscopy. Filamentous EcGS is induced by environmental metal ions. We used cryo-electron microscopy (cryo-EM) to decipher the structure of metal ion (nickel)-induced EcGS helical filament at a sub-3Å resolution. EcGS filament formation involves stacking of native d ...[more]

PMID: 35481643

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Project description:Previous transcriptomic studies have shown that glnA, which codes for glutamine synthetase (GS), is induced during growth of uropathogenic E. coli in urine. GS is at the intersection of carbon and nitrogen metabolism and is, not surprisingly, highly regulated. The glnALG operon specifies GS and two regulators of the nitrogen-regulated (Ntr) response, which scavenges nitrogenous compounds during nitrogen limitation. Transcription of glnA initiates from the cAMP receptor protein (Crp)-dependent glnAp1 and the GlnG-dependent glnAp2 promoters which respond to carbon and nitrogen limitation, respectively. High glnA expression is most often associated with nitrogen limitation, and its expression in urine could suggest nitrogen limitation, despite a high concentration of ammonia which suppresses glnA expression. To understand the basis for this expression, we compared growth of a non-pathogenic and a uropathogenic strain of E. coli in minimal media and a urine-like medium in wild-type, ΔglnG and Δcrp strains and assessed glnA expression with transcriptional and translational fusions, quantitative reverse transcription PCR, direct enzymatic assay, and Western blots. Our results showed that urea induced expression from the Crp-dependent glnAp1 promoter which produced a transcript that was not translated, inhibited expression from the Ntr glnAp2 promoter, and did not induce other Ntr genes, i.e., high glnA transcription can occur without induction of the Ntr response. We conclude that urea induces glnA expression in E. coli that is independent of the regulators of the Ntr response.IMPORTANCEThe bacteria that cause urinary tract infections often become resistant to antibiotic treatment, and genes expressed during an infection could suggest non-antibiotic targets. During growth in urine, glnA (specifying glutamine synthetase) expression is high, but our results show that urea induces glnA expression independent of the regulation that responds to nitrogen limitation. Although our results suggest that glnA is an unlikely target for therapy because of variation in urinary components between individuals, our analysis of glnA expression in urine-like environments has revealed previously undescribed layers of regulation. In other words, regulatory mechanisms that are discovered in a laboratory environment do not necessarily operate in the same way in nature.

Dataset Information

Structural basis for the helical filament formation of Escherichia coli glutamine synthetase.

Publications

Structural basis for the helical filament formation of Escherichia coli glutamine synthetase.

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