Solution structure of the Pseudomonas putida protein PpPutA45 and its DNA complex

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Proline utilization A (PutA) is a multifunctional enzyme in gram-negative bacteria, catalyzing proline oxidation and acting as a cytoplasmic repressor.
• In Pseudomonas putida, PutA regulates gene expression when proline concentrations are low.

Purpose of the Study:

• To elucidate the structural basis of DNA binding and dimerization for the N-terminal domain of P. putida PutA (PpPutA45).
• To understand the mechanism of DNA sequence recognition by PpPutA45.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to determine the solution structure of PpPutA45.
• DNA gel mobility shift assays and NMR chemical shift perturbations (CSPs) for DNA binding studies.
• Haddock 2.1 for molecular modeling of the PpPutA45-DNA complex.

Main Results:

• PpPutA45 forms a symmetrical homodimer (12 kDa) with a ribbon-helix-helix (RHH) structure.
• PpPutA45 specifically binds a 14 base-pair DNA oligomer.
• The dimeric structure features an antiparallel beta-sheet that inserts into the DNA major groove, with key residues (Thr5, Gly7, Lys9) mediating sequence-specific contacts.

Conclusions:

• The N-terminal RHH domain of PutA is responsible for DNA binding and dimerization.
• The dimeric structure and specific amino acid residues are crucial for recognizing and binding DNA, likely playing a role in PutA's auto-repression function.
• Conserved RHH domains suggest conserved interdomain interactions in PutA evolution.