Cyanobacteria are phototrophic prokaryotes that evolved oxygenic photosynthesis ∼2.7 billion y ago and are presently responsible for ∼10% of total global photosynthetic production. To cope with the evolutionary pressure of dropping ambient CO concentrations, they evolved a CO-concentrating mechanism (CCM) to augment intracellular inorganic carbon (C) levels for efficient CO fixation. However, how cyanobacteria sense the fluctuation in C is poorly understood. Here we present biochemical, structural, and physiological insights into SbtB, a unique P-like signaling protein, which provides new insights into C sensing. SbtB is highly conserved in cyanobacteria and is coexpressed with CCM genes. The SbtB protein from the cyanobacterium sp. PCC 6803 bound a variety of adenosine nucleotides, including the second messenger cAMP. Cocrystal structures unraveled the individual binding modes of trimeric SbtB with AMP and cAMP. The nucleotide-binding pocket is located between the subunit clefts of SbtB, perfectly matching the structure of canonical P proteins. This clearly indicates that proteins of the P superfamily arose from a common ancestor, whose structurally conserved nucleotide-binding pocket has evolved to sense different adenyl nucleotides for various signaling functions. Moreover, we provide physiological and biochemical evidence for the involvement of SbtB in C acclimation. Collectively, our results suggest that SbtB acts as a C sensor protein via cAMP binding, highlighting an evolutionarily conserved role for cAMP in signaling the cellular carbon status.

The application of state-of-the-art deep-learning approaches to the protein modeling problem has expanded the "high-accuracy" category in CASP14 to encompass all targets. Building on the metrics used for high-accuracy assessment in previous CASPs, we evaluated the performance of all groups that submitted models for at least 10 targets across all difficulty classes, and judged the usefulness of those produced by AlphaFold2 (AF2) as molecular replacement search models with AMPLE. Driven by the qualitative diversity of the targets submitted to CASP, we also introduce DipDiff as a new measure for the improvement in backbone geometry provided by a model versus available templates. Although a large leap in high-accuracy is seen due to AF2, the second-best method in CASP14 out-performed the best in CASP13, illustrating the role of community-based benchmarking in the development and evolution of the protein structure prediction field.

Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to the OB rings.

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