Mechanism for Nucleotidyl Transfer in LINE-1 ORF2p Revealed by QM/MM Simulations

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Long Interspersed Element-1 (L1) retrotransposons are significant components of the human genome.
• ORF2p, a key enzyme in L1 retrotransposition, possesses endonuclease and reverse transcriptase activities.
• Existing structural data show a single Mg2+ ion in the reverse transcriptase active site, differing from typical DNA polymerase mechanisms.

Purpose of the Study:

• To investigate the DNA elongation mechanism of the L1 retrotransposon ORF2p.
• To elucidate the role of metal ions in the reverse transcriptase active site.
• To reconcile structural findings with enzymatic activity.

Main Methods:

• Combined quantum mechanics (QM) and molecular mechanics (MM) simulations using PBE0-D3/6-31G** and CHARMM.
• Biased umbrella sampling molecular dynamics simulations.
• Umbrella integration to calculate the free energy profile of the nucleotidyl transfer reaction.

Main Results:

• The DNA elongation reaction proceeds via a single-step nucleotidyl transfer.
• A free energy barrier of 15.1 ± 0.8 kcal/mol was determined for the reaction.
• Product stabilization was calculated at 7.8 ± 1.2 kcal/mol.
• The mechanism involves a concerted nucleophilic attack and proton transfer without a second catalytic metal ion.
• The estimated rate constant of ~60 s-1 is consistent with known RT kinetics.

Conclusions:

• The L1 ORF2p reverse transcriptase utilizes a distinct catalytic mechanism involving a single Mg2+ ion.
• The reaction proceeds through a dissociative mechanism, challenging the canonical two-metal-ion model for DNA polymerases.