GInK, a PII-homologue:: Structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition

GlnK is a recently discovered homologue of the P-II signal protein, an indicator of the nitrogen status of bacteria. P-II occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it. is known that GlnK behaves Like P-II and can substitute for P-II under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of P-II is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and P-II. Clearly, the structural similarities and differences of GlnK and P-II will lead to a better understanding of how P-II-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 Angstrom resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to P-II but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the P-II structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 3(10) helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes; Go-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and Is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence-is more highly conserved than the functional T-loop, suggesting that ATP has an important role in P-II-like proteins. (C) 1998 Academic Press.