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PII signalling proteins constitute one of the largest families of signalling proteins in nature. An even larger superfamily of trimeric sensory proteins with the same architectural principle as PII proteins appears in protein structure databases. Large surface-exposed flexible loops protrude from the intersubunit faces, where effector molecules are bound that tune the conformation of the loops. Via this mechanism, PII proteins control target proteins in response to cellular ATP/ ADP levels and the 2-oxoglutarate status, thereby coordinating the cellular carbon/nitrogen balance. The antagonistic ( ATP versus ADP) and synergistic (2-oxoglutarate and ATP) mode of effector molecule binding is further affected by PII-receptor interaction, leading to a highly sophisticated signalling network organized by PII. Altogether, it appears that PII is a multitasking information processor that, depending on its interaction environment, differentially transmits information on the energy status and the cellular 2-oxoglutarate level. In addition to the basic mode of PII function, several bacterial PII proteins may transmit a signal of the cellular glutamine status via covalent modification. Remarkably, during the evolution of plant chloroplasts, glutamine signalling by PII proteins was re-established by acquisition of a short sequence extension at the C-terminus. This plant-specific C-terminus makes the interaction of plant PII proteins with one of its targets, the arginine biosynthetic enzyme N-acetyl-glutamate kinase, glutamine-dependent. [ABSTRACT FROM AUTHOR]

PII proteins constitute a superfamily of highly conserved signaling devices, common in all domains of life. Through binding of the metabolites ATP, ADP and 2-oxoglutarate (2-OG), they undergo conformational changes which allow them to regulate a variety of target proteins including enzymes, transport proteins and transcription factors. But, in reverse, these target proteins also modulate the metabolite sensing properties of PII, as has been recently shown. We used this effect to refine our PII based Förster resonance energy transfer (FRET) sensor and amplify its sensitivity towards ADP. With this enhanced sensor setup we addressed the question whether the PII protein from the model organism Synechococcus elongatus autonomously switches into the ADP conformation through ATPase activity as proposed in a recently published model. The present study disproves ATPase activity as a relevant mechanism for the transition of PII into the ADP state. In the absence of 2-OG, only the ATP/ADP ratio and concentration of ADP directs the competitive interaction of PII with two targets, one of which preferentially binds PII in the ATP-state, the other in the ADP-state. [ABSTRACT FROM AUTHOR]

PII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048-1059. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction. [ABSTRACT FROM AUTHOR]

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml -1 , a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

PII protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many PII proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, PII protein from Synechococcus elongatus, a model for cyanobacteria and plant PII proteins, is utilized to reveal the anticooperative mechanism upon binding of 2-oxoglutarate (2-OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2-OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T-loop, which is believed to be responsible for mediating the binding of PII with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of PII variants further confirmed the influence of T-loop on the anticooperative regulation, especially on binding of the third 2-OG. These discoveries extend our understanding of the PII T-loop from being essential in versatile binding of target protein to signal-mediating in the anticooperative allosteric regulation., (© 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.)