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 structure of the bacterial signal transduction protein

The Escherichia coli signal transduction protein P,,, 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 P,, with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6, with a = b = 6 1.6 A, c = 56.3 A and V,,, 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 f 1,000 for P,, in solution. This result is consistent with the native protein being a homotrimer.

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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.

The crystalline environment affects the relative orientation of the two B-bane! domains of bovine pancreatic B-trypsin (BPT) and induces conformational strain. The comparison is based on anisotropic refinement using SHELXL-93 [I] of BPT at "5°C in two different orthorhombic crystal forms [2, 3] at 1.0-l.l A resolution (R=8.3%; 9.8%). The accuracy in the atomic positions is on average 0.01-0.02 A for each structural model. When aligned qn the active site region and one domain (150 residues; rmsd 0.09 A, max. deviation 0.19 A for the main chain atoms), the other domain exhibits relative coordinate shifts (nnsd 0.22 A, max. 0.52 A) as well as substantial changes in the conformational angles. The positional and confom1ational differences are particularly large for the B-strands 81-90 (near the surface) and 104-108. Molecular packing interactions further induce flexibility in a number of residues (35 in the one structure, 24 in the other) that are present in discrete altemate conformations. Conelations between altemate side chain locations are observed which extend over distances up to 20 A; in several cases, water or sulphate molecules with partial occupancies are involved. Most of the ordered solvent (ca. 2.2 waters per accessible residue-nearly all in the first coordination shells) and the degree of anisotropy in the individual atomic temperature factors are essentially not affected by the crystalline environment. In both structures, only one residue (Gin 192) is not located in well-defined electron density; the flexibility may reflect its functional role in orienting substrates. The diffraction data were measured on the synchrotron beamline BW6/DORIS.

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