Structure And Regulation Of The Key Proteins To The Heterocyst Development Of Anabaena Sp. PCC 7120

Cyanobacteria are prokaryotes, also known as"blue-green algae", which can execute photosynthesis. Some cyanobacteria can also fix nitrogen, such as the filamental cyanobacterium Anabaena, which live in the fresh water. When the nutrition is abundant, there are vegetative cells along the filament. While the envoriment is short of combined nitrogen, some vegetative cells will develop into heterocysts, usually every other 10 vegetative cells. These heterocysts can fix nitrogen, by transforming it into a combined form and transporting the fixed nitrogen to the neighbor vegetative cells, so that the whole filament can survive from the nitrogen starvation. These characteristics make Anabaena an excellent model for studying the cell differentiation.2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed to be a putative receptor of 2-OG. NtcA belongs to the Crp-Fnr family, which is featured by CAP (cAMP-activated catabolite activator protein). Here we present three crystal structures of NtcA from the cyanobacterium Anabaena sp. strain PCC 7120: the apo-form (1.90 (A|°)), and two ligand-bound forms in complex with either 2-OG (2.60 (A|°)) or its analogue 2,2-difluoropentanedioic acid (2.40 (A|°)). All structures assemble as homodimers, with each subunit composed of an N-terminal effector binding domain (EBD) and a C-terminal DNA binding domain (DBD) connected by a long helix (C-helix). The 2-OG binds to the EBD at a pocket similar to that used by cAMP in CAP (cAMP-activated catabolite activator protein), but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. While CAP adopts a transition from off to on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apo form, how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms. P_Ⅱproteins are highly conserved signal transducers in bacteria, archaea and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors, to regulate the functions of diverse protein partners. The P_Ⅱpartner PipX, first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of P_Ⅱand NtcA by PipX in a 2-OG-dependent manner enables P_Ⅱto indirectly regulate the transcriptional activity of NtcA. We solved the crystal structure of the P_Ⅱ–PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 (A|°) resolution. A homotrimeric P_Ⅱcaptures three subunits of PipX through the T-loops. Similar to P_Ⅱfrom Synechococcus, the core structure consists of an antiparallelβ-sheet with fourβ-strands and twoα-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallelβ-strands and twoα-helices, which is reminiscent of the P_Ⅱstructure. The T-loop of each P_Ⅱsubunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between theβ-sheets of PipX and P_Ⅱcore structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial P_Ⅱproteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA–PipX complex. Taken together, these findings provide structural insights into how P_Ⅱregulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.This study fucosed on the proteins involved in the early stage of heterocyst development and these output results provided structural insights for the regulatory mechanism of heterocyst development.