Increased ionic strength triggers multiple conformations in both apo and holo forms of bacterial ferric binding protein

This study combines molecular dynamics (MD) simulations with solution X-ray scattering measurements to investigate the range of conformations that can be achieved by a pH/ionic strength (IS) sensitive protein and to quantify its distinct populations. To explore how the conformational multiplicity of proteins might be modified in the environmental niches of biological media, we focus on the periplasmic protein FbpA from H. influenzae which is a part of the mechanism developed by bacteria to capture iron from higher organisms. We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its unique biological environment. The collected SAXS data complemented by SEC analyses and binding assays point to multiple conformations at physiological IS while they are well-explained by single x-ray structures in a 15 mM buffer. Moreover, by fitting the SAXS data with unique conformations sampled by a series of MD simulations carried out under conditions mimicking the buffers, we quantify with high accuracy the populations of the occupied substates. Furthermore, we find the D52A mutant that we predicted by coarse-grained computational approaches to allosterically control the iron binding site in FbpA responds to the environmental changes in our experiments with varied conformational selection scenarios. We show an application of the environment dependence of the conformers by designing a genetically encoded iron biosensor as a chimera of FbpA and green fluorescent protein using the D52 region as the insertion point. This work exemplifies how unifying a range of experimental and computational techniques provides platforms for achieving the next generation of applications that put protein dynamics rather than the static structure at the centerstage. Support by Turkish Atomic Energy Authority, TUBITAK grant no 121Z329 and a scholarship to GL through 2214-A program. Measurements conducted at EMBL-Hamburg.