Structural insights into HetR-PatS interaction involved in cyanobacterial pattern formation

Abstract

The one-dimensional pattern of heterocyst in the model cyanobacterium Anabaena sp. PCC 7120 is coordinated by the transcription factor HetR and PatS peptide. Here we report the complex structures of HetR binding to DNA, and its hood domain (HetRHood) binding to a PatS-derived hexapeptide (PatS6) at 2.80 and 2.10 Å, respectively. The intertwined HetR dimer possesses a couple of novel HTH motifs, each of which consists of two canonical α-helices in the DNA-binding domain and an auxiliary α-helix from the flap domain of the neighboring subunit. Two PatS6 peptides bind to the lateral clefts of HetRHood, and trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary α-helix and eventually release of HetR from the DNA major grove. These findings provide the structural insights into a prokaryotic example of Turing model.

Figure 1. Schematic diagram of the signaling cascade of heterocyst development and pattern formation.

Figure 2. Structure of HetR−DNA complex.

(a) Overall structure of Anabaena HetR dimer in complex with the 21-bp DNA from the hetP promoter. The secondary structural elements of DBD (limon), flap domain (red) and hood domain (pink) in subunit A are labeled. The missing residues are depicted as the dashed lines. Comparison of DNA-bound Anabaena HetR (cyan) with (b) the DNA-bound (wheat) and (c) apo-form (purple) Fischerella HetR. The rotation angles of the flap domains are labeled. (d) Schematic representation of interaction networks between Anabaena HetR and DNA. Interacting residues are marked with the same color as their corresponding domains, respectively. Direct hydrogen bonds are indicated as black lines and water-mediated hydrogen bonds as blue dashes. (e) EMSA assays of HetR with variations of DNA. DNA samples with and without HetR are shown as “+” and “−”, respectively. The corresponding DNA variations numbered as No.1 to 7. The 5′-g⁶gg and 5′-c¹⁴cc motifs are highlighted in dark orange.

Figure 3. PatS6-binding mode.

(a) Overall structure of the PatS6−HetR_Hood dimer. Two PatS6 peptides (yellow) are shown as sticks, with the F_o − F_c electron-density omit map contoured at 3.0 sigma. (b) A close-up view of the PatS6-binding site. Hydrogen bonds and the polar interactions are indicated as dashed lines. (c) EMSA assays of HetR and mutants with original DNA sequence, in the presence or absence of PatS6. “−” represents DNA probe without protein. “+” indicates DNA incubated with HetR or mutants, whereas “S” indicates addition of PatS6 to the HetR−DNA complex. (d) The distance between Arg223 and the C-terminus of PatS6 in each subunit is labeled.

Figure 4. Putative conformational changes of HetR upon PatS6 binding.

Computational simulation of (a) HetR−DNA and (b) PatS6−HetR−DNA. The structure ensembles, including DNA (light orange) and PatS6 (yellow), are presented as ribbon. (c) Fluorescence lifetime spectroscopy assays of six HetR mutants. The amplitude-averaged lifetimes (τ_AV) of six HetR mutants with standard error are shown as histograms. (d) The relative changes of τ_AV in percentage. The data are analyzed using an independent samples t-test (**stands for p < 0.01).

Figure 5. A proposed inhibition mechanism of HetR upon binding to PatS.

The duplex DNA is shown as a cartoon, and PatS as a yellow sphere. The HTH motifs, including the two auxiliary α10 helices, are shown as cylinders.