Structural basis for the helical filament formation of <i>Escherichia coli</i> glutamine synthetase

Huang, Pei‐Chi; Chen, Shao‐Kang; Chiang, Wei-Hung; Ho, Meng‐Ru; Wu, Kuen‐Phon

doi:10.1002/pro.4304

Cited by 8 publications

(7 citation statements)

References 41 publications

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“…Helical reconstruction was carefully performed following the common RELION protocol 49,52 and our previous work. 53 The initial helical rise at 4.8 Å was calculated from the 2D class layer line profiles. The sinogram initial model was generated using selected 2D class averages, whereas the crossover distance, helical rise, and helical twist were 450 Å, 4.8 Å, and −1.8°, respectively.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108–144 Fibril Reveals an Ordered Water Channel in the Center

et al. 2022

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Fibrils of the hamster prion peptide (sHaPrP, sequence 108–144) were prepared in an acidic solution, and their structure was solved by cryogenic electron microscopy with a resolution of 2.23 Å based on the gold-standard Fourier shell correlation (FSC) curve. The fibril has a novel architecture that has never been found in other amyloid fibrils. Each fibril is assembled by four protofilaments (PFs) and has an ordered water channel in the center. Each protofilament contains three β-strands (125–130, 133–135, and 138–141) arranged in an “R”-shaped construct. The structural data indicate that these three β-strand segments are the most amyloidogenic region of the prion peptide/protein and might be the site of nucleation during fibrillization under conditions without denaturants.

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Section: ■ Results and Discussionmentioning

confidence: 99%

“…Approximately 138 000 2D particles were selected after few runs of 2D classifications. Helical reconstruction was carefully performed following the common RELION protocol , and our previous work . The initial helical rise at 4.8 Å was calculated from the 2D class layer line profiles.…”

Section: Methodsmentioning

confidence: 99%

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108–144 Fibril Reveals an Ordered Water Channel in the Center

et al. 2022

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“…Finally, higher oligomeric states of GS enzymes have been known for a long time for organisms like yeast and E. coli (He et al, 2009; Huang et al, 2022; Petrovska et al, 2014; Valentine et al, 1968). Interestingly, dependent on the ice thickness and on higher concentrated areas of the grids, we could also observe filament-like structures of M. mazei GlnA 1 in cryo-EM and resolved their structure at a resolution of 6.9 Å (suppl.…”

Section: Discussionmentioning

confidence: 99%

Section: -Og Is Crucially Required For M Mazei Gs Assembly To An Acti...mentioning

confidence: 99%

2-oxoglutarate triggers assembly of active dodecamericMethanosarcina mazeiglutamine synthetase

Herdering,

Reif-Trauttmansdorff,

Kumar

et al. 2024

Preprint

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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeonMethanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1and sP26) has been reported. Here, we show that the strong activation ofM. mazeiGS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from monomers/dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of the transition state catalytic conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on an allosteric binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction ofM. mazeiGS into a catalytically active dodecamer is not affected by GlnK1and crucially depends on the presence of 2-OG.

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“…Why these enzymes form polymeric filaments and/or cellular self-assemblies remains an important question. Some hypotheses include rapid activation or inactivation of enzymatic activity, modulation of allosteric responses, storage and protection from autophagy, control of cellular localization, and/or buffering of enzyme activity in cells. , While the structures of these filamentous enzymes are now being revealed, − including for the SgrAI system, − far fewer studies have been performed to determine the underlying kinetic mechanisms of the phenomenon.…”

Section: Introductionmentioning

confidence: 99%

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease

Ghadirian,

Morgan,

Horton

2024

Biochemistry

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Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

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

Cited by 8 publications

References 41 publications

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108–144 Fibril Reveals an Ordered Water Channel in the Center

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108–144 Fibril Reveals an Ordered Water Channel in the Center

2-oxoglutarate triggers assembly of active dodecamericMethanosarcina mazeiglutamine synthetase

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease

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