Structural and Functional Characterization of PII and PII-like Proteins and their Network of Interactions
Year: 2020
Type: dissertation
Abstract: The signaling transduction proteins of PII superfamily are one of the ancient and highly conserved protein families in nature and widely distributed in all domains of life. Canonical PII exerts it’s signaling function via the binding of small effector molecules, including ATP, ADP, and 2-oxoglutarate (2-OG). All cyanobacteria contain a PII homologue of the GlnB-type, which is involved in the regulation of nitrogen assimilation by binding to N-acetyl-L-glutamate kinase (NAGK), the key enzyme of arginine biosynthesis, and to PII-interacting protein X (PipX), the co-activator of the global nitrogen-trascription factor NtcA. The binding of PII to NAGK enhances NAGK activity and prevents arginine (Arg) feedback inhibition, thereby increasing the flux into the arginine pathway. Formation of the PII-PipX complex causes a deactivation of the NtcA regulon. In this study, we identified Lys58 to be a key residue for proper sensing/signaling function of cyanobacterial PII protein. Furthermore, this study provided the first biochemical characterization of red algal PII protein from Porphyra purpurea. Its properties represent an intermediary state between PII properties in Cyanobacteria and Chlorophyta. A comparison to Chlorophyta PII proteins showed that during later stages of evolution of Chlorophyta, the PII proteins diverged in their properties, becoming very heterogeneous with respect to ADP and to 2-OG binding, while only the binding of ATP in presence of 2-OG has been conserved. Further, we characterized the first metabolic adaptation strategies of colorless-nonphotosynthetic alga Polytomella parva, in comparison to closely related green-photosynthetic alga Chlamydomonas reinhardtii, in response to nitrogen starvation. Metabolome analysis revealed that P. parva accumulates higher amounts of TCA intermediates as well as arginine, glutamate and aspartate. Strikingly, the PII and NAGK proteins in P. parva have coevolved into a stable hetero-oligomeric complex, irrespective of effector molecules such that PII evolved into a subunit of NAGK. The PII-like proteins lack the canonical PII signature sequences but are structurally clearly related to classical PII proteins. Their functions, targets and regulatory responses are unknown. A close examination of available cyanobacteria genomes revealed several different PII-like proteins. In this study, we focused on two PII-like proteins in cyanobacteria namely: 1) SbtB protein, which is located in one operon next to sodium dependent bicarbonate transporter (SbtA) and 2) the divalent ion tolerance protein CutA. Biochemical and physiological studies showed that the SbtB protein is an important component of the cyanobacterial carbon concentration mechanism (CCM) for sensing the fluctuation levels of inorganic carbon (Ci) via binding to the secondary messenger cAMP. Also, we demonstrated that cyanobacterial CutA protein is not involved directly in heavy metal sensing. SbtB was revealed as the first protein of the PII superfamily, which specifically binds cAMP in addition to ATP, ADP and AMP. However, for CutA, no binding of effector molecules could be identified. I resolved the crystal structures of CutA (in apo form) and SbtB (in apo status and in complex with AMP and cAMP) proteins. The nucleotide-binding pocket of SbtB is located between the lateral cleft of two subunits, as in canonical PII proteins. Apparently, the trimeric core architectural principle of PII-like proteins of CutA and SbtB are widely distributed similar to the canonical PII core architecture. This clearly indicates that the proteins of the PII superfamily arose evolutionary from a common trimeric ancestor protein.
Source: MPG.PuRe (Max Planck Society)
Author: Selim, Khaled
Language: English
Subfield: Plant Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3094616298