Mutations in the WFYY motif alter human PrimPol activity: structural insights from atomistic MD simulations

Human PrimPol (hPrimPol) is the primase and polymerase enzyme involved in reinitiating DNA replication at stalled replication forks. The hPrimPol mutants are linked to multiple ophthalmoplegic conditions and mitochondriopathies. The WFYY motif of hPrimPol was reported to play an essential role in stabilizing the incoming dNTP. Earlier experimental studies reported that the W87G and Y90D mutations significantly reduced the primase and polymerase activities of hPrimPol. To elucidate the structural implications of these single-point mutations for polymerase activity, we have performed microsecond molecular dynamics simulations of the wild-type and mutant complexes W87G, F88L, Y89D, and Y90D. Analyses focused on the conserved residues W87 and Y90, whose mutations abolish polymerase activity, enabling us to compare their structural and dynamic changes relative to the WT complex. Significant changes in the structural parameters essential for successful replication and the residue interaction network were observed in the inactive mutants. Community analysis revealed that amino acid residues 280-300 fluctuate in W87G and Y90D, leading to disorientation of the incoming dCTP and changes in the motion of the ModC module. Additionally, intra-module communication between ModN and ModC is altered, which affects the binding affinity of dCTP and, consequently, leads to a loss of polymerase activity. Overall, our studies rationalize the experimental results and provide further insights into the structural effects of single-point mutations in the WFYY motif on hPrimPol activity and their implications in harnessing this motif as an allosteric module for drug targeting.