Crystallization and Preliminary X-ray Diffraction Studies of New Crystal Forms of<i>Escherichia coli</i>P<sub>II</sub>Complexed with Various Ligands

Edwards, Karen J.; Suffolk, P. M.; Carr, Pamela; Wegman, M.; Cheah, Eong; Ollis, David L.

doi:10.1107/s0907444996003241

Cited by 2 publications

(3 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The form II crystals were obtained at 277 K with the protein concentrated to 18–25 mg·mL −1 and stored in buffer A with 20 m m spermidine and a fourfold molar excess of ATP and α‐KG. The form II crystals were prepared from a solution that contained sodium acetate, poly(ethylene glycol) 3400 and Hepes pH 7.5 as described previously [18]. The space group was I222.…”

Section: Methodsmentioning

confidence: 99%

“…Data for both crystal forms were processed using the hkl program [19] and the results are shown in Table 1. Data for the form II crystals were collected at 100 K as described previously [18]. The form III crystals were mounted in glass capillaries and data were collected at 293 K. The first data set (form IIIa) was collected with a crystal that had been transferred to the reservoir buffer of the crystallization experiment while the second (form IIIb) was collected with a crystal soaked in 10 m m ATP, 10 m m CaCl 2 , 10% isopropanol and 100 m m sodium acetate pH 4.7.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The structure of the P_II–ATP complex

Carr

Huber

et al. 2001

European Journal of Biochemistry

View full text Add to dashboard Cite

P II is a signal transduction protein that is part of the cellular machinery used by many bacteria to regulate the activity of glutamine synthetase and the transcription of its gene. The structure of P II was solved using a hexagonal crystal form (form I). The more physiologically relevant form of P II is a complex with small molecule effectors. We describe the structure of P II with ATP obtained by analysis of two different crystal forms (forms II and III) that were obtained by co-crystallization of P II with ATP. Both structures have a disordered recognition (T) loop and show differences at their C termini. Comparison of these structures with the form I protein reveals changes that occur on binding ATP. Surprisingly, the structure of the P II /ATP complex differs with that of GlnK, a functional homologue. The two proteins bind the base and sugar of ATP in a similar manner but show differences in the way that they interact with the phosphates. The differences in structure could account for the differences in their activities, and these have been attributed to a difference in sequence at position 82. It has been demonstrated recently that P II and GlnK form functional heterotrimers in vivo. We construct models of the heterotrimers and examine the junction between the subunits.Keywords: ATP binding protein; GlnK; P II ; signal transduction; X-ray structure.P II is a signal transduction protein found in a variety of organisms. It is best characterized in Escherichia coli in which it indicates the nitrogen status of the cell. In many bacteria the level of available nitrogen is closely coupled to the level of glutamine synthetase (GS) activity. P II is involved in the regulation of GS and the transcription of its gene [1±4]. In brief, the nitrogen status of the cell is monitored by the uridylyl transferase/uridylyl removing enzyme (UT/URase). It uridylylates P II at Tyr51 to form P II ±UMP [5±7] under conditions of limiting ammonia and catalyses the removal of UMP under conditions of excess ammonia. The uridylylation status of P II reflects the nitrogen status of the cell. P II dictates the direction of the enzymatic regulation of GS through its interaction with adenylyl transferase (ATase). The transcriptional regulation of GS is mediated by P II and the nitrogen regulatory proteins I and II (NRI and NRII) also known as NtrC and NtrB, respectively. Although the regulation of GS has been studied for many years, much remains to be learnt about this system in the light of some recent discoveries. For example, it is now apparent that many organisms possess two P II -like proteins. The second protein in E. coli is known as GlnK [8,9]. The physical properties of GlnK are very similar to those of P II and under some circumstances GlnK can substitute for P II . When the cell is grown in nitrogen-poor conditions, P II and GlnK form heterotrimers [10] to fine-tune the regulation of GS [11]. The fine regulation of the nitrogen signal cascade by functional heterotrimers of P II and GlnK introduces a new level of complexity to ...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The structure of the P_II–ATP complex

Carr

Huber

et al. 2001

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…A second, independent test of the need for uridylylation of GlnK to relieve NifL inhibition investigated the effects of a mutant form of GlnK in which the presumed site of uridylylation, Y51 (1,11,13,23,36,50), was altered. Western blot analysis ( Fig.…”

Section: Compare Strain Ncm2087 [Glnk Mutant Strain With Pjes1104]mentioning

confidence: 99%

Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions

Soupène

Ninfa

et al. 1998

J Bacteriol

View full text Add to dashboard Cite

In Klebsiella pneumoniae, NifA-dependent transcription of nitrogen fixation (nif) genes is inhibited by a flavoprotein, NifL, in the presence of molecular oxygen and/or combined nitrogen. We recently demonstrated that the general nitrogen regulator NtrC is required to relieve NifL inhibition under nitrogen (N)-limiting conditions. We provide evidence that the sole basis for the NtrC requirement is its role as an activator of transcription for glnK, which encodes a P II -like allosteric effector. Relief of NifL inhibition is a unique physiological function for GlnK in that the structurally related GlnB protein of enteric bacteria-apparently a paralogue of GlnK-cannot substitute. Unexpectedly, although covalent modification of GlnK by uridylylation normally occurs under N-limiting conditions, several lines of evidence indicate that uridylylation is not required for relief of NifL inhibition. When GlnK was synthesized constitutively from non-NtrC-dependent promoters, it was able to relieve NifL inhibition in the absence of uridylyltransferase, the product of the glnD gene, and under N excess conditions. Moreover, an altered form of GlnK, GlnK Y51N , which cannot be uridylylated due to the absence of the requisite tyrosine, was still able to relieve NifL inhibition.

show abstract

Crystallization and Preliminary X-ray Diffraction Studies of New Crystal Forms ofEscherichia coliP_IIComplexed with Various Ligands

Cited by 2 publications

References 7 publications

The structure of the P_II–ATP complex

The structure of the P_II–ATP complex

Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions

Contact Info

Product

Resources

About

Crystallization and Preliminary X-ray Diffraction Studies of New Crystal Forms ofEscherichia coliPIIComplexed with Various Ligands

Cited by 2 publications

References 7 publications

The structure of the PII–ATP complex

The structure of the PII–ATP complex

Physiological Role for the GlnK Protein of Enteric Bacteria: Relief of NifL Inhibition under Nitrogen-Limiting Conditions

Contact Info

Product

Resources

About

Crystallization and Preliminary X-ray Diffraction Studies of New Crystal Forms ofEscherichia coliP_IIComplexed with Various Ligands

The structure of the P_II–ATP complex

The structure of the P_II–ATP complex