Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV

Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae. •Optimized Purification: Overcame challenges in CpendoIV purification by strategically manipulating NaCl concentrations, enhancing overall protein yield and purity.•Crystallographic Breakthrough: Successful crystallization of CpendoIV at 1.91 Å resolution provides a crystallographic platform for in-depth structural studies.•Innovative Functional Analysis: Revolutionizing endonuclease study with a non-traditional approach, prioritizing DNA binding dynamics over cleavage activity.•Mutational Insight for Drug Development: Mutagenesis (A75, A108, G109) unveils crucial DNA binding residues, providing a roadmap for targeted drug development.•Unveiling DNA-Protein Interaction: Shifting focus from enzymatic activity to binding dynamics, this research reshapes endonuclease investigation paradigms.