AlkD’s Conformational Dynamics Regulated by Protein–DNA Interactions for Effective Target Recognition

<i>Bacillus cereus</i>alkylpurine DNA glycosylase AlkD plays a critical role in preserving genomic integrity by selectively excising cytotoxic positively charged lesions. Unlike most DNA glycosylases, AlkD employs a unique non-base-flipping mechanism involving distinct conformational states for lesion searching and excision. However, the interplay between its conformational dynamics and lesion recognition remains unclear. This study combines microsecond-scale molecular dynamics (MD) simulations, scanning fluorescence resonance energy transfer-fluorescence correlation spectroscopy (FRET-FCS) experiments, and cellular assays to investigate the lesion recognition mechanism mediated by the AlkD-dsDNA complex. We identified two critical residues, W109 and R148, that act as molecular probes to recognize DNA lesions and mismatches. These residues alter the equilibrium between the search complex (SC) and excision complex (EC), primarily distinguished by their dsDNA conformations. W109 and R148 exhibit enhanced recognition capabilities when the lesion is positively charged, explaining AlkD's selectivity toward such lesions. Together, our study has identified the critical residues for recognizing the lesion and mismatch and regulating the enzyme's conformational dynamics, providing valuable molecular insights into the target search and lesion recognition of AlkD.