An Investigation of the Conformational Dynamics of ABC Exporter PCAT1 using Microsecond-Level MD Simulations

Area of Science:

• Structural Biology and Biochemistry
• Molecular Biophysics
• Membrane Transport Proteins

Background:

• Peptidase-containing ATP-binding cassette transporters (PCATs) are crucial for bacterial secretion, coupling ATP hydrolysis to peptide processing and export.
• The precise molecular mechanisms governing nucleotide binding and stabilization in PCATs, particularly PCAT1, are not fully understood.
• PCAT1 may exhibit distinct nucleotide preferences compared to canonical ATP-binding cassette (ABC) transporters.

Purpose of the Study:

• To characterize the nucleotide binding, protein stability, and conformational dynamics of PCAT1 using advanced computational methods.
• To elucidate the roles of Mg2+ and substrate peptides in stabilizing PCAT1 conformations and nucleotide interactions.
• To identify specific residues and molecular interactions responsible for nucleotide stabilization within the PCAT1 transporter.

Main Methods:

• Microsecond-scale all-atom molecular dynamics (MD) simulations of PCAT1 in various states (inward-facing/outward-facing, with/without Mg2+ and substrate).
• Free energy perturbation (FEP) calculations to thermodynamically analyze nucleotide binding affinities.
• Residue-level free energy decomposition to pinpoint individual residue contributions to nucleotide binding energetics.

Main Results:

• Substrate and Mg2+ cooperatively stabilize the inward-facing (IF) conformation of PCAT1, reducing fluctuations and enhancing nucleotide retention.
• Absence of Mg2+ leads to increased nucleotide mobility and partial dissociation, highlighting its critical role in stabilization.
• FEP calculations confirm strong ATP stabilization in the IF state with Mg2+, while Lys525 in the Walker A motif is identified as a key residue for dominant stabilizing interactions.

Conclusions:

• Mg2+ coordination and substrate binding are essential for stabilizing the PCAT1 IF state and organizing the nucleotide-binding site for efficient ATP hydrolysis.
• The study provides a detailed molecular and energetic description of nucleotide recognition and stabilization in PCAT1.
• Residue-level free energy analysis is a powerful approach for dissecting the energetic landscape of nucleotide binding in ABC transporters.