Eong Cheah

Structure of the Escherichia coli signal transducing protein PII.

BACKGROUND In Gram-negative proteobacteria, the nitrogen level in the cell is reflected by the uridylylation status of a key signal transducing protein, PII. PII modulates the activity of glutamine synthetase (GS) through its interaction with adenylyl transferase and it represses the expression of GS by acting in concert with nitrogen regulatory protein II. RESULTS The three-dimensional structure of the Escherichia coli PII trimer has been determined at 2.7 A resolution. PII shows a low level of structural similarity to a broad family of alpha/beta proteins and contains a double beta alpha beta motif. The PII trimer contains three beta-sheets, each of which is composed of strands from each of the three monomers. These are surrounded by six alpha-helices. CONCLUSIONS The structure of PII suggests potential regions of interaction with other proteins and serves as an initial step in understanding its signal transducing role in nitrogen regulation.

The role of the T-loop of the signal transducing protein PII from Escherichia coli

The 3D structure of PII, the central protein that controls the level of transcription and the enzymatic activity of glutamine synthetase in enteric bacteria revealed that residues 37–55 form the ‘T’ loop, part of which protrudes from the core of the protein. Within this loop are the only two tyrosine residues that occur in the polypeptide, and one of them, Tyr‐51, has been shown by chemical modification studies to be the site of uridylylation. Since tyrosine at position 46 is conserved in all known PII, proteins, oligonucleotide directed mutagenesis was used to investigate the role of the two residues. Changing Tyr‐51 to phenylalanine or serine abolished uridylylation. Altering tyrosine at position 46 to phenylalanine affected the rate of uridylylation of the protein. This latter mutation does not alter the structure of PII but the reduction in the uridylylation efficiency suggests a role for this residue in recognition and binding of the sensor enzyme uridylyl transferase.

X-ray structure of the signal transduction protein from Escherichia coli at 1.9 A.

The structure of the bacterial signal transduction protein P(II) has been refined to an R factor of 13.2% using 3sigma data between 10 and 1.9 A. The crystals exhibited twinning by merohedry and X-ray intensities were corrected using the method of Fisher & Sweet [Fisher & Sweet (1980). Acta Cryst. A36, 755-760] prior to refinement. Our earlier 2.7 A structure [Cheah, Carr, Suffolk, Vasudevan, Dixon & Ollis (1994). Structure, 2, 981-990] served as a starting model. P(II) is a trimeric molecule, each subunit has a mass of 12.4 kDa and contains 112 amino-acid residues. The refined model includes all 1065 protein atoms per subunit plus 312 water molecules. The high-resolution refinement confirms the correctness of our 2.7 A model, although it leads to a redefinition of the extent of various secondary-structural elements. The monomeric structure of P(II) exhibits an interlocking double betaalphabeta fold. This is a stable fold found in a number of proteins with diverse functions. The association of the protein into a trimer leads to a new structure which we describe in detail. The effects of crystal packing forces are discussed and potential interaction sites with other proteins and effector molecules are identified.

Escherichia coli PII protein: purification, crystallization and oligomeric structure

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix‐assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 × 0.2 × 0.3 mm were grown and analysed by X‐ray diffraction. The crystals belong to space group P63 with a=b=61.6Å,c= 56.3 Å and V m of 2.5 for one subunit in the asymmetric unit. A low‐resolution electron density map showed electron density concentrated around a three‐fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 ± 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

Crystallization and preliminary X-ray analysis of Escherichia coli GlnK.

The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.

Crystallization and preliminary X-ray analysis of IND, an enzyme with indole oxygenase activity from Chromobacterium violaceum.

IND, a redox flavoprotein from Chromobacterium violaceum has been crystallized in the presence and absence of NADH. The crystals belong to the space group P41212 or its enantiomorph P43212 with a = 73.9 and c = 153.6 A. There is one molecule per asymmetric unit and the crystals diffract beyond 2.1 A resolution.

Crystallization and Preliminary X-ray Diffraction Studies of New Crystal Forms of Escherichia coli PII Complexed with Various Ligands

New crystals of the signal-transducing protein P(II) have been obtained in the presence of a number of different effector ligands. Various crystal forms are observed depending on the nature of the ligand(s). Co-crystallization with 2-ketoglutarate, glutamate and pyrophosphate produces hexagonal crystals similar to the wild type, ATP yields cubic crystals and ATP in conjunction with 2-ketoglutarate or glutamate yields orthorhombic crystal forms. All of the above crystals have been characterized by X-ray diffraction analysis. The hexagonal crystals belong to space group P6(3), cubic crystals to either I23 or I2(1)3 and orthorhombic crystals to I222. A molecular-replacement solution for the P(II)/ATP/2-ketoglutarate crystals has been obtained giving us an initial model for a trimer in the orthorhombic crystal form.