Perturbation-Response Scanning Reveals Ligand Entry-Exit Mechanisms of Ferric Binding Protein

Abstract

We study apo and holo forms of the bacterial ferric binding protein (FBP) which exhibits the so-called ferric transport dilemma: it uptakes iron from the host with remarkable affinity, yet releases it with ease in the cytoplasm for subsequent use. The observations fit the “conformational selection” model whereby the existence of a weakly populated, higher energy conformation that is stabilized in the presence of the ligand is proposed. We introduce a new tool that we term perturbation-response scanning (PRS) for the analysis of remote control strategies utilized. The approach relies on the systematic use of computational perturbation/response techniques based on linear response theory, by sequentially applying directed forces on single-residues along the chain and recording the resulting relative changes in the residue coordinates. We further obtain closed-form expressions for the magnitude and the directionality of the response. Using PRS, we study the ligand release mechanisms of FBP and support the findings by molecular dynamics simulations. We find that the residue-by-residue displacements between the apo and the holo forms, as determined from the X-ray structures, are faithfully reproduced by perturbations applied on the majority of the residues of the apo form. However, once the stabilizing ligand (Fe) is integrated to the system in holo FBP, perturbing only a few select residues successfully reproduces the experimental displacements. Thus, iron uptake by FBP is a favored process in the fluctuating environment of the protein, whereas iron release is controlled by mechanisms including chelation and allostery. The directional analysis that we implement in the PRS methodology implicates the latter mechanism by leading to a few distant, charged, and exposed loop residues. Upon perturbing these, irrespective of the direction of the operating forces, we find that the cap residues involved in iron release are made to operate coherently, facilitating release of the ion.

Author Summary

Upon binding ligands, many proteins undergo structural changes compared to the unbound form. We introduce a methodology to monitor these changes and to study which mechanisms arrange conformational shifts between the liganded and free forms. Our method is simple, yet it efficiently characterizes the response of proteins to a given perturbation on systematically selected residues. The coherent responses predicted are validated by molecular dynamics simulations. The results indicate that the iron uptake by the ferric binding protein is favorable in a thermally fluctuating environment, while release of iron is allosterically moderated. Since ferric binding protein exhibits a high sequence identity with human transferrin whose allosteric anion binding sites generate large conformational changes around the binding region, we suggest mutational studies on remotely controlling sites identified in this work.