How conformational dynamics of DNA polymerase select correct substrates: experiments and simulations

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Enzymes undergo significant structural changes upon substrate binding.
• Understanding these conformational dynamics is key to enzyme function, particularly in fidelity mechanisms.
• HIV reverse transcriptase is a critical target for antiviral therapies, making its substrate selection process important.

Purpose of the Study:

• To investigate the role of structural changes in HIV reverse transcriptase during substrate selection.
• To elucidate the mechanism by which the enzyme distinguishes between correct and incorrect nucleotides.
• To correlate experimental observations with theoretical predictions of conformational dynamics.

Main Methods:

• Utilized atomically detailed simulations employing the Milestoning method.
• Performed experimental analyses to validate simulation predictions.
• Analyzed free energy profiles and reaction coordinates of conformational changes.

Main Results:

• Simulations accurately predicted the rate and free energy profile of conformational changes.
• A millisecond timescale conformational change was identified that locks correct nucleotides and releases incorrect ones.
• Early events like weak substrate binding and protein conformational transitions, rather than the chemical step, dictate reaction yield.

Conclusions:

• Conformational changes are critical for the fidelity of HIV reverse transcriptase.
• The enzyme's ability to select correct substrates is established in the initial binding and conformational transition phases.
• These findings offer insights into enzyme mechanisms and potential therapeutic strategies targeting HIV replication.