P _II -like signaling protein SbtB links cAMP sensing with cyanobacterial inorganic carbon response

Abstract

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.

Structural basis for the allosteric regulation of the SbtA bicarbonate transporter by the PII-like protein, SbtB, fromCyanobiumsp. PCC7001

“…While the interaction between the a-phosphate and Arg43 could be possible when AMP or ADP is bound, it seems that the additional ATP-specific interaction with Arg46 is required to first stabilize this basal T-loop region and promote this interaction between the a-phosphate and Arg43. Thus, in comparison to the largely disordered T-loop residues 43-55 in apo-SbtB7001, AMP:SbtB7001, ADP:SbtB7001 and cAMP:SbtB7001 (as was also observed in structures of SbtB6803 bound to AMP and cAMP [10]), the binding of ATP to SbtB7001 resulted in stabilization of the T-loop. These structural observations are consistent with our ITC experiments for ATP, which revealed a substantially greater entropic cost associated with ATP binding, compared with the binding of AMP, ADP and cAMP ( Table 1).…”

“…In this work we have shown that, like SbtB6803 [10], SbtB7001 binds AMP, ADP, cAMP and ATP with micromolar-range affinities. However, in contrast to SbtB6803, we found that SbtB7001 binds ATP with an affinity 5-to 10-fold greater than the other nucleotides tested, and that the affinity for ATP increases a further 10-fold ( transition from light to darkness in cyanobacteria [35].…”

“…Selim et al [10] recently reported that SbtB6803 binds AMP, ADP, cyclic-AMP (cAMP) and ATP with micromolar-range affinities, although the reported range of K D values was relatively narrow µM from microscale thermophoresis experiments, 75-250 µM for K D3 from ITC experiments). AMP and ADP were observed to stabilize the SbtA-SbtB6803 complex in vivo, and the authors suggested that the cAMP:AMP ratio may act as a signal to control SbtA6803mediated HCO 3transport in vivo.…”

Interaction of N‐acetyl‐l‐glutamate kinase with the PII signal transducer in the non‐photosynthetic alga Polytomella parva: Co‐evolution towards a hetero‐oligomeric enzyme

“…The PII homologues (GlnB and GlnK), which contain the conserved PROSITE motifs (PTM‐site: PS00496 and C‐terminal signature: PS00638) , are referred as canonical PII proteins (reviewed in ). The PII members, which demonstrate the same trimeric architectural principle as GlnB/GlnK proteins but lack their typical PROSITE signature pattern, are termed as the PII‐like proteins .…”

A novel Ca2+-binding protein influences photosynthetic electron transport inAnabaenasp. PCC 7120

“…Isothermal titration calorimetry (ITC) was performed in Tris-HCl buffer (pH 7.9), using a VP-ITC microcalorimeter (MicroCal) as described [34] after extensive dialysis of the purified recombinant protein in Chelex 100 (Sigma) to remove contaminating divalent cations. Calorimetric data were evaluated using MicroCal software (OriginLab) and fitted into one-site and two sequential binding sites models, for calculation of the binding thermodynamics [35]. ITC runs were repeated at least three times with two different batches of purified recombinant protein.…”