Dynamics and Conformations of a Full-Length CRESS-DNA Replicase.

Area of Science:

• Structural biology and virology
• Molecular dynamics simulations of viral proteins

Background:

• Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses utilize a Replicase (Rep) protein for replication.
• Rep is a helicase with endonuclease, oligomeric, and ATPase domains (ED, OD, AD).
• Previous cryo-EM studies revealed PCV2 Rep structure, hexamer formation, ssDNA/nucleotide binding, and AD staircase arrangement, suggesting a hand-over-hand translocation mechanism.

Purpose of the Study:

• To scrutinize the proposed hand-over-hand mechanism of ssDNA translocation by the ATPase domain (AD) of CRESS-DNA Rep.
• To investigate the dynamics of Rep in different states using all-atom Molecular Dynamics (MD) simulations.
• To understand the role of ssDNA and ADP in stabilizing the AD staircase arrangement.

Main Methods:

• All-atom Molecular Dynamics (MD) simulations of the PCV2 Rep protein.
• Simulations were conducted for three conditions: Rep with ssDNA and ADP, Rep with ssDNA, and Rep alone.
• Each simulation ran for 700 nanoseconds, with convergence observed within 200 nanoseconds.

Main Results:

• MD simulations provided insights into the dynamics of Rep and its interactions with ssDNA and ADP.
• The simulations demonstrated the importance of ssDNA and ADP in driving the AD to adopt the staircase conformation.
• This study represents the first all-atom MD simulation of a CRESS-DNA Rep protein.

Conclusions:

• The study validates the dynamic behavior of Rep and its components under different binding conditions.
• Findings support the proposed hand-over-hand mechanism for ssDNA translocation, driven by nucleotide binding and hydrolysis.
• This work establishes a foundation for future MD studies to elucidate the chemical mechanisms of Rep-mediated DNA translocation.