Ligand-induced conformation changes drive ATP hydrolysis and function in SMARCAL1

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Mutations and deletions in SMARCAL1, an SWI2/SNF2 protein, cause Schimke immuno-osseous dysplasia (SIOD). SMARCAL1 preferentially binds to DNA molecules possessing double-stranded to single-stranded transition regions and mediates annealing helicase activity. The protein is critical for alleviating replication stress and maintaining genome integrity. In this study, we have analysed the ATPase activity of three mutations – A468P, I548N and S579L – present in SIOD patients. These mutations are present in RecA-like domain I of the protein. Analysis using active DNA-dependent ATPase A domain (ADAAD), an N-terminal deleted construct of bovine SMARCAL1, showed that all three mutants were unable to hydrolyse ATP. Conformational studies indicated that the α-helix and β-sheet content of the mutant proteins was altered compared to the wild-type protein. Molecular simulation studies confirmed that major structural changes had occurred in the mutant proteins. These changes included alteration of a loop region connecting motif Ia and II. As motif Ia has been implicated in DNA binding, ligand binding studies were done using fluorescence spectroscopy. These studies revealed that the Kd for protein–DNA interaction in the presence of ATP was indeed altered in the case of mutant proteins compared to the wild-type. Finally, in vivo studies were done to complement the in vitro and in silico studies. The results from these experiments demonstrate that mutations in human SMARCAL1 that result in loss in ATPase activity lead to increased replication stress and therefore possibly manifestation of SIOD.SMARCAL1, a DNA-dependent ATPase, undergoes a conformation change in the presence of ATP that allows DNA to bind resulting in ATP hydrolysis. Mutations in SMARCAL1 cause major structural changes in the protein leading to impaired DNA binding in the presence of ATP, resulting in the loss of ATPase activity.

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