Structural Insights Into The DNA-binding And Inactivation Of Cyanobacterial Transcription Factor Hetr Involved In Heterocyst Development

Cyanobacteria, also known as blue-green algae, have been living on the Earth for more than 3 billion years. Cyanobacteria are a large group of Gram-negative prokaryotes that perform oxygenic photosynthesis via vegetative cells. Over the course of evolution, cyanobacteria have provided the major source of O2 in the atmosphere and hence are principally responsible for the Great Oxygenation Event. Besides, the photosynthetic features of cyanobacteria are similar to those found in green plant chloroplasts, organelles whose evolutionary ancestors are probably considered to be cyanobacteria.Biological nitrogen fixation is mediated by the nitrogenase system that catalyses the ATP dependent reduction of atmospheric N2 to ammonia. A variety of prokaryotic organisms can fix N2, including Clostridium pasteurianum, Azotobacter chroococcum, and some filamentous cyanobacteria. Except for vegetative cells, cyanobacteria have evolved multiple specialized cell types, including nitrogen-fixing heterocysts, spore-like akinetes, and the cells of motile hormogonia filaments. In response to combined nitrogen starvation, about 5-10% of the vegetative cells of particular filamentous cyanobacteria, such as the model cyanobacterium Anabaena sp. PCC 7120, can differentiate into heterocysts to fix N2. Along the filaments, heterocysts are regularly intercalated and usually separated by 10-20 vegetative cells. This one-dimensional regular arrangement of heterocysts is called heterocyst pattern.Usually the whole process of heterocysts development may take about 20-24 hr. The heterocyst is morphologically distinguishable from the neighboring vegetative cells in larger size, two neck-shaped ends and a thicker envelope. The envelop of heterocyst contains two additional layers:a glycolipid layer that reduces the permeation of oxygen and a polysaccharide layer that protects the fragile glycolipid layer. Mature heterocysts do not have the O2-evolving photosystem II. Besides, heterocysts display an increased respiration rate to further decrease the oxygen content in the cell. As the heterocysts become microaerobic, N2 fixation is initiated, and active growth of the filament is resumed.The Kreb’s cycle in cyanobacteria is incomplete because of the absence of the enzyme 2-oxoglutarate dehydrogenase. The deprivation of combined nitrogen leads to the accumulation of 2-oxogluatarate, which in consequence activates the global nitrogen regulator NtcA, followed by the increased expression of HetR, to initiate the differentiation of heterocysts. HetR has long been recognized as a major player in the regulation of heterocyst development and patterning. A hetR-null mutant of Anabaena sp. PCC 7120 is unable to initiate heterocyst differentiation, whereas various hetR site-mutants either abolish, reduce or greatly enhance heterocyst differentiation. In practice, HetR acts as a transcription factor to initiate a signaling cascade that ultimately is responsible for the activation of multiple downstream genes. HetR dimer specifically binds to a consensus DNA sequence at the promoter regions of hetP, hetZ, hetR, patS, pknE, hepA, etc.The inhibitory pentapeptide RGSGR (PatS5) is involved in heterocysts pattern formation. This motif was first identified as the last five residues of PatS, and later within HetN. PatS and HetN are negative regulators of heterocyst development. Binding of PatS5 to HetR may abolish the DNA-binding activity of HetR. Notably, HetR has a higher affinity towards a hexapeptide ERGSGR (PatS6), compared to that of PatS5. The network between HetR and the diffusible inhibitory peptide in heterocyst patterning conforms to the basis of morphygens described for Turing model in developmental biology. However, the bona fide active form of the inhibitory peptide under physiological conditions remains unknown.Here we performed the studies on the structures and functions of several key proteins involved in heterocyst development. Finally, we solved the complex structures of HetR binding to DNA, and its hood domain (HetRHood) binding to PatS6 at 2.80 and 2.10 A, respectively. In the HetR-DNA complex, the intertwined HetR dimer possesses a couple of novel DNA-binding motifs, each of which consists of a canonical helix-turn-helix (HTH) motif and an auxiliary a-helix from the flap domain of the neighboring subunit. In the PatS6-HetRHood complex, two PatS6 peptides bind to the lateral clefts of HetRHood. Further study showed that binding of PatS6 to HetR may trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary a-helix and eventually release of HetR from the DNA major grove. Later, we performed the experiments on the study of HetN and HetF. We purified the recombinated HetN protein, but failed on get the crystals. We expressed and purified the N and C-domian of HetF, respectively; however, only the C-domain of HetF was crystallized, which is under optimization.