Dottorato in BIOCHIMICA

Titolo della tesi: Cryo-EM structure of PdxR from Bacillus clausii in complex with its target DNA.

PdxR is a bacterial transcription factor belonging to the MocR family, which is involved in the regulation of the de novo biosynthesis of pyridoxal 5’-phosphate (PLP). PdxR has been extensively studied at the functional level and its target regulon has been characterized. However, a structural characterization of this transcription factor, and of MocRs in general, is missing. In fact, to date no structure of MocRs in complex with DNA is available, and a mechanism of their regulatory activity has been hypothesised on the basis of functional and computational data. In this thesis I report the structure of PdxR from Bacillus clausii in complex with its target DNA sequence of 48-bp, determined by cryo-electron microscopy (cryo-EM). Our data revealed that in PdxR the DNA-binding winged helix-turn-helix domain (wHTH) and the aspartate aminotransferase (AAT)-like domain are arranged in a domain-swap homodimeric assembly and are connected by a long flexible linker. Single particle analysis allowed us to isolate two different conformational states of the PdxR-DNA complex, an “open” and a “closed” one, differently bound to the target oligonucleotide, and to identify residues on the protein and bases on the DNA involved in sequence recognition and complex formation. Moreover, we analysed the role of both the DNA sequence and conformation in the specific recognition by PdxR, evaluating the complex stability using rationally designed DNA sequences. Our results provide relevant information on the structure and the dynamics of PdxR-DNA complex and represent a fundamental step to clarify the mechanism governing the DNA-binding mode and the transcriptional regulation of MocRs transcription factors.