Effects of T-loop modification on the PII-signalling protein: structure of uridylylated Escherichia coli GlnB bound to ATP.

To adapt to environments with variable nitrogen sources and richness, the widely distributed homotrimeric PII signalling proteins bind their allosteric effectors ADP/ATP/2-oxoglutarate, and experience nitrogen-sensitive uridylylation of their flexible T-loops at Tyr51, regulating their interactions with effector proteins. To clarify whether uridylylation triggers a given T-loop conformation, we determined the crystal structure of the classical paradigm of PII protein, Escherichia coli GlnB (EcGlnB), in fully uridylylated form (EcGlnB-UMP3 ). This is the first structure of a postranslationally modified PII protein. This required recombinant production and purification of the uridylylating enzyme GlnD and its use for full uridylylation of large amounts of recombinantly produced pure EcGlnB. Unlike crystalline non-uridylylated EcGlnB, in which T-loops are fixed, uridylylation rendered the T-loop highly mobile because of loss of contacts mediated by Tyr51, with concomitant abolition of T-loop anchoring via Arg38 on the ATP site. This site was occupied by ATP, providing the first, long-sought snapshot of the EcGlnB-ATP complex, connecting ATP binding with T-loop changes. Inferences are made on the mechanisms of PII selectivity for ATP and of PII-UMP3 signalling, proposing a model for the architecture of the complex of EcGlnB-UMP3 with the uridylylation-sensitive PII target ATase (which adenylylates/deadenylylates glutamine synthetase [GS]) and with GS.

Structure of AmtR, the global nitrogen regulator of Corynebacterium glutamicum, in free and DNA-bound forms.

UNLABELLED: Corynebacterium glutamicum is a bacterium used for industrial amino acid production, and understanding its metabolic pathway regulation is of high biotechnological interest. Here, we report crystal structures of AmtR, the global nitrogen regulator of C. glutamicum, in apo (2.25-Å and 2.65-Å resolution) and DNA-bound (3-Å resolution) forms. These structures reveal an all-α homodimeric TetR family regulator composed of a helix-turn-helix-hosting N-terminal DNA-binding domain and a C-terminal dimerization domain. AmtR has several unique structural features that appear to be invariant among AmtR proteins, which may be related to its regulation by the nitrogen-sensing trimeric protein GlnK rather than by small-molecule effectors. As compared with other TetR family members, AmtR has an extra C-terminal helix, a large extended external loop that resembles the flexible tranducer T-loop of GlnK in sequence, and a large open cavity towards the intersubunit region that changes shape upon DNA binding. The marked kinking of helix 4 decreases in the DNA-bound form. The binding of one AmtR dimer to its DNA operator involves not only the insertion of helices 3 and 3' in adjacent turns of the double-helix major groove, but also the anchoring of 19-residue, arginine-rich and proline-rich N-terminal extensions to two external minor grooves. Electrophoretic mobility shift assays with a deletion mutant reveal that the 19-residue extension is crucial for AmtR binding to DNA. N-extension anchoring explains the flanking by AT sequences of the recognized target DNA sequence core. The significance of these findings for the entire TetR family of regulators and for GlnK regulation of AmtR is discussed. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 5DXZ (native AmtR), 5DY1 (SeMet-AmtR), and 5DY0 (AmtR·DNA)].

The structure of a PII signaling protein from a halophilic archaeon reveals novel traits and high-salt adaptations.

To obtain insights into archaeal nitrogen signaling and haloadaptation of the nitrogen/carbon/energy-signaling protein PII, we determined crystal structures of recombinantly produced GlnK2 from the extreme halophilic archaeon Haloferax mediterranei, complexed with AMP or with the PII effectors ADP or ATP, at respective resolutions of 1.49 Å, 1.45 Å, and 2.60 Å. A unique trait of these structures was a three-tongued crown protruding from the trimer body convex side, formed by an 11-residue, N-terminal, highly acidic extension that is absent from structurally studied PII proteins. This extension substantially contributed to the very low pI value, which is a haloadaptive trait of H. mediterranei GlnK2, and participated in hexamer-forming contacts in one crystal. Similar acidic N-extensions are shown here to be common among PII proteins from halophilic organisms. Additional haloadaptive traits prominently represented in H. mediterranei GlnK2 are a very high ratio of small residues to large hydrophobic aliphatic residues, and the highest ratio of polar to nonpolar exposed surface for any structurally characterized PII protein. The presence of a dense hydration layer in the region between the three T-loops might also be a haloadaptation. Other unique findings revealed by the GlnK2 structure that might have functional relevance are: the adoption by its T-loop of a three-turn α-helical conformation, perhaps related to the ability of GlnK2 to directly interact with glutamine synthetase; and the firm binding of AMP, confirmed by biochemical binding studies with ATP, ADP, and AMP, raising the possibility that AMP could be an important PII effector, at least in archaea. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession numbers 4OZL (hmGlnK2-AMP), 4OZJ (hmGlnK2-ADP), and 4OZN (hmGlnK2-ATP). STRUCTURED DIGITAL ABSTRACT: hmGlnK2 and hmGlnK2 bind by x-ray crystallography (View interaction).

Aequorin-expressing yeast emits light under electric control.

In this study, we show the use of direct external electrical stimulation of a jellyfish luminescent calcium-activated protein, aequorin, expressed in a transgenic yeast strain. Yeast cultures were electrically stimulated through two electrodes coupled to a standard power generator. Even low (1.5 V) electric pulses triggered a rapid light peak and serial light pulses were obtained after electric pulses were applied periodically, suggesting that the system is re-enacted after a short refraction time. These results open up a new scenario, in the very interphase between synthetic biology and cybernetics, in which complex cellular behavior might be subjected to electrical control.