Expanding the nitrogen regulatory protein superfamily: Homology detection at below random sequence identity.

Nitrogen regulatory (PII) proteins are signal transduction molecules involved in controlling nitrogen metabolism in prokaryots. PII proteins integrate the signals of intracellular nitrogen and carbon status into the control of enzymes involved in nitrogen assimilation. Using elaborate sequence similarity detection schemes, we show that five clusters of orthologs (COGs) and several small divergent protein groups belong to the PII superfamily and predict their structure to be a (betaalphabeta)(2) ferredoxin-like fold. Proteins from the newly emerged PII superfamily are present in all major phylogenetic lineages. The PII homologs are quite diverse, with below random (as low as 1%) pairwise sequence identities between some members of distant groups. Despite this sequence diversity, evidence suggests that the different subfamilies retain the PII trimeric structure important for ligand-binding site formation and maintain a conservation of conservations at residue positions important for PII function. Because most of the orthologous groups within the PII superfamily are composed entirely of hypothetical proteins, our remote homology-based structure prediction provides the only information about them. Analogous to structural genomics efforts, such prediction gives clues to the biological roles of these proteins and allows us to hypothesize about locations of functional sites on model structures or rationalize about available experimental information. For instance, conserved residues in one of the families map in close proximity to each other on PII structure, allowing for a possible metal-binding site in the proteins coded by the locus known to affect sensitivity to divalent metal ions. Presented analysis pushes the limits of sequence similarity searches and exemplifies one of the extreme cases of reliable sequence-based structure prediction. In conjunction with structural genomics efforts to shed light on protein function, our strategies make it possible to detect homology between highly diverse sequences and are aimed at understanding the most remote evolutionary connections in the protein world.

Kinch LN ，Grishin NV 《-》

GlnK, 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 PII signal protein, an indicator of the nitrogen status of bacteria. PII 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 PII and can substitute for PII 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 PII 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 PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A 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 PII 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 PII 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 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-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 PII-like proteins.

Xu Y ，Cheah E ，Carr PD ，van Heeswijk WC ，Westerhoff HV ，Vasudevan SG ，Ollis DL ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Gleaning non-trivial structural, functional and evolutionary information about proteins by iterative database searches.

Using a number of diverse protein families as test cases, we investigate the ability of the recently developed iterative sequence database search method, PSI-BLAST, to identify subtle relationships between proteins that originally have been deemed detectable only at the level of structure-structure comparison. We show that PSI-BLAST can detect many, though not all, of such relationships, but the success critically depends on the optimal choice of the query sequence used to initiate the search. Generally, there is a correlation between the diversity of the sequences detected in the first pass of database screening and the ability of a given query to detect subtle relationships in subsequent iterations. Accordingly, a thorough analysis of protein superfamilies at the sequence level is necessary in order to maximize the chances of gleaning non-trivial structural and functional inferences, as opposed to a single search, initiated, for example, with the sequence of a protein whose structure is available. This strategy is illustrated by several findings, each of which involves an unexpected structural prediction: (i) a number of previously undetected proteins with the HSP70-actin fold are identified, including a highly conserved and nearly ubiquitous family of metal-dependent proteases (typified by bacterial O-sialoglycoprotease) that represent an adaptation of this fold to a new type of enzymatic activity; (ii) we show that, contrary to the previous conclusions, ATP-dependent and NAD-dependent DNA ligases are confidently predicted to possess the same fold; (iii) the C-terminal domain of 3-phosphoglycerate dehydrogenase, which binds serine and is involved in allosteric regulation of the enzyme activity, is shown to typify a new superfamily of ligand-binding, regulatory domains found primarily in enzymes and regulators of amino acid and purine metabolism; (iv) the immunoglobulin-like DNA-binding domain previously identified in the structures of transcription factors NFkappaB and NFAT is shown to be a member of a distinct superfamily of intracellular and extracellular domains with the immunoglobulin fold; and (v) the Rag-2 subunit of the V-D-J recombinase is shown to contain a kelch-type beta-propeller domain which rules out its evolutionary relationship with bacterial transposases.

Aravind L ，Koonin EV 《JOURNAL OF MOLECULAR BIOLOGY》

Sensory components controlling bacterial nitrogen assimilation.

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits information on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.

Kamberov ES ，Atkinson MR ，Feng J ，Chandran P ，Ninfa AJ ... -  《-》

Detection of 3D atomic similarities and their use in the discrimination of small molecule protein-binding sites.

Najmanovich R ，Kurbatova N ，Thornton J 《-》