Conformational multiplicity of bacterial ferric binding protein revealed by small angle x-ray scattering and molecular dynamics calculations

Abstract

This study combines molecular dynamics(MD) simulations with small angle X-ray scattering(SAXS) measurements to investigate range of conformations that can be adopted by a pH/ionic strength(IS) sensitive protein and to quantify its distinct populations in solution.To explore how conformational distribution of proteins may be modified in the environmental niches of biological media,we focus on the periplasmic ferric binding protein A(FbpA) from H.influenzae involved in the mechanism by which bacteria capture iron from higher organisms.We examine iron-binding/release mechanisms of FbpA in varying conditions simulating its biological environment.While we show that these changes fall within the detectable range for SAXS as evidenced by differences observed in the theoretical scattering patterns calculated from the crystal structure models of apo and holo forms,detection of conformational changes due to point mutation D52A and changes in ionic strength(IS) from SAXS scattering profiles have been challenging.Here, statistical analyses with SAXS profiles and results from different techniques were combined. SAXS data complemented by size exclusion chromatography(SEC) and iron-binding assays point to multiple and/or alternative conformations at physiological IS whereas they are well-explained by single crystallographic structures in low IS buffers.By fitting the SAXS data with unique conformations sampled by a series of MD simulations under conditions mimicking the buffers, we quantify the populations of the occupied substates.We also find that the D52A mutant that we predicted by coarse-grained computational modeling to allosterically control the iron binding site in FbpA, responds to the environmental changes in our experiments with conformational selection scenarios that differ from those of the wild type.

Allosteric modulation of fluorescence revealed by hydrogen bond dynamics in a genetically encoded maltose biosensor

“…SAXS experiments. 24 On the downside, it is not straightforward to deduce ON and OFF states of the conformations obtained from MD simulations which are based on classical physics, while the proton transfer reaction requires very costly quantum chemistry calculations. 5,25 It is therefore essential to derive descriptors for conformations obtained from MD simulations to characterize the ON state of a GEFB.…”

“…20 Such dynamical approaches are also effective in predicting the role of environmental conditions such as pH and ionic strength 21,22 and are essential for interpreting the conformational multiplicity of proteins detected by, for example, SAXS experiments. 23 On the downside, it is not straightforward to deduce ON and OFF states of the conformations obtained from MD simulations which are based on classical physics, while the proton transfer reaction requires very costly quantum chemistry calculations. 5,24 It is therefore essential to derive descriptors for conformations obtained from MD simulations to characterize the ON state of a GEFB.…”

“…For example, a recent report revealed the working mechanism of a single FP based cAMP biosensor by using metadynamics molecular dynamics (MD) simulations to sample the apo form starting from the holo crystal structure 20 . Such dynamical approaches are also effective in predicting the role of environmental conditions such as pH and ionic strength 21,22 and are essential for interpreting the conformational multiplicity of proteins detected by, for example, SAXS experiments 23 . On the downside, it is not straightforward to deduce ON and OFF states of the conformations obtained from MD simulations which are based on classical physics, while the proton transfer reaction requires very costly quantum chemistry calculations 5,24 .…”