Protein control of a ligand: Modeling nitric oxide release in nitrophorin 4

Abstract

The nitric oxide (NO) transport protein nitrophorin 4 (Np4) is able to modulate NO release rates by two orders of magnitude in response to pH change, and the rates are much slower than in the classic transport protein myoglobin. Experiments have shown that a large conformational change in two loops near the heme binding site from a closed state at pH 5 to an open pocket at pH 7 is apparently responsible for controlling NO release. The mechanism of protein control of ligand escape was investigated using atomic-resolution X-ray crystallography, molecular dynamics simulations, and stochastic modeling. Crystal structures at pH 5 and pH 7 with and without NO revealed that the loops exhibit a mixture of conformations under all the conditions, suggesting they are not a static barrier to NO escape. Molecular dynamics simulations at both pH 5 and pH 7 were performed to observe NO migration as a function of protein conformation. The simulations agree closely with the X-ray structures, and show the loops opening and NO escaping at pH 7, while at pH 5 the loops remain closed and NO never leaves the binding pocket. A stochastic model was based on these observations, modeling the loops as a gate fluctuating between open and closed states and NO as a diffusing particle inside the protein, where it can rebind to the heme or escape out of the protein. An analytical solution of NO escape rates as a function of loop opening and closing rates demonstrates that in the appropriate regime the escape rate is determined by the probability of ligand rebinding to the heme. This indicates the reason for the difference in off rates between Np4 and Mb: Np4 encourages rebinding, while Mb provides internal space for ligand migration. Similarly, pH dependence of Np4 off rates is attributed to a greater rebinding fraction in the closed state at pH 5. However, the source of multiple rates in the experimental kinetics remains unclear, and the model will need to be extended to capture this complexity.