Investigating the Dynamic Binding Behavior of PMX53 Cooperating with Allosteric Antagonist NDT9513727 to C5a Anaphylatoxin Chemotactic Receptor 1 through Gaussian Accelerated Molecular Dynamics and Free-Energy Perturbation Simulations.

C5a anaphylatoxin chemotactic receptor 1 (C5aR1) is an important target in anti-inflammatory therapeutics. The cyclic peptide antagonist PMX53 binds to the orthosteric site located in the extracellular vestibule of C5aR1, and the non-peptide antagonist NDT9513727 binds to the allosteric site formed by the middle region of TM3 (trans-membrane helix), TM4, and TM5. We catch a sight of the variational binding mode of PMX53 during the Gaussian accelerated molecular dynamic (GaMD) simulations. In the binary complex of C5aR1 and PMX53, the PMX53 takes a dynamic binding mechanism during the simulation. Namely, the side chain of Arg 6 of PMX53 extends to TM6-TM7 (pose 1) or swings to TM5 (pose 2), forming a salt bridge with Glu199. Meanwhile, in the ternary complex of C5aR1 with PMX53 and NDT9513727, the side chain of Arg 6 of PMX53 swings to TM5 (pose 2) from extending to TM6-TM7 (pose 1) at the beginning of the GaMD simulation. In subsequent simulation, PMX53 stabilizes in the pose 2 binding mode by forming a stable salt bridge with Glu199. The free-energy perturbation (FEP) calculations demonstrate that pose 1 (Δ G binding = -10.94 kcal/mol) is more stable in the binary complex and pose 2 (Δ G binding = -7.91 kcal/mol) is unstable because of highly dynamic TM5. NDT9513727 interacts directly with TM4 and TM5 and stabilizes the hydrophobic stack between the extracellular sides of the two helices. Therefore, pose 2 (Δ G binding = -16.27 kcal/mol) is notably stable than pose 1 (Δ G binding = -9.78 kcal/mol) in the ternary complex. The identification of a novel binding mode of PMX53 and the detailed structural information of PMX53 interacting with a receptor obtained by GaMD simulations will be helpful in designing potent antagonists of C5aR1.