Snapshots of Motion: A Novel Structural Intermediate Reveals Conserved Dynamics in Archaeal DNA Ligases

We present the first x-ray crystallographic structural evidence of an archaeal DNA ligase showing the AMP covalent adduct together with further cofactor hydrolysis, capturing a transient intermediary in the first step of the ligation reaction, triggered by the pyrophosphate hydrolysis. Our crystallographic models of Thermococcus gammatolerans DNA ligase (LigTgam), coupled with bioinformatic analysis of at least 28 crystallographic structures from ATP- and NAD⁺-dependent DNA ligases, highlight the central role of domain mobility. Notably, elevated B-values are consistently observed in key catalytic and binding regions, suggesting a link between structural flexibility and enzymatic efficiency. Remarkably, this pattern of high B-values is conserved in replicative ligases, including bacterial Lig A, indicating a broader evolutionary relevance. These fluctuations emphasize the importance of conformational adaptability in accommodating substrate DNA and facilitating catalytic steps, including adenylation and phosphodiester bond formation. In this work, we delve deeper into this dynamic behavior, providing evidence of its critical role in ligase function.