Characterizing the conformational dynamics of metal-free PsaA using molecular dynamics simulations and electron paramagnetic resonance spectroscopy

Abstract

After the embargo period via non-commercial hosting platforms such as their institutional repository  via commercial sites with which Elsevier has an agreement In all cases accepted manuscripts should: link to the formal publication via its DOI  bear a CC-BY-NC-ND license -this is easy to do, click here to find out how  if aggregated with other manuscripts, for example in a repository or other site, be shared in alignment with our hosting policy  not be added to or enhanced in any way to appear more like, or to substitute for, the published journal article unknown. Here, we use continuous wave electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations to study the relative flexibility of the structural domains in metal-free PsaA and its distribution of conformations in solution. The results show that the crystal structure of the metal-free PsaA is a good representation of the dominant conformation in solution, but the protein also samples structurally distinct conformations that are not captured by the crystal structure. Further, these results suggest that the metal binding site is larger and more solvent exposed than indicated by the metal-free crystal structure. Collectively, this study provides atomicresolution insight into the conformational dynamics of PsaA prior to metal binding and lays the groundwork for future EPR and MD based studies of PsaA in solution. 4 GRAPHICAL ABSTRACT HIGHLIGHTS• PsaA is the Mn 2+ -recruiting protein of the human pathogen Streptococcus pneumoniae• Metal-free PsA samples structurally distinct conformations not captured by the crystal structure• The metal binding site is larger and more solvent exposed than indicated by the crystal structure KEYWORDSCluster A-I solute-binding protein (SBP); Streptococcus pneumoniae; PsaA; molecular dynamics simulations, electron paramagnetic resonance (EPR) spectroscopy; protein flexibility; spin-label mobility 5

Pneumococcal lipoproteins involved in bacterial fitness, virulence, and immune evasion

“…In crystals, the protein folds into a two‐lobed structure that consists of an N‐ and C‐terminal domain providing the metal‐binding site and linked by a rigid α‐helix which has been proposed to restrict conformational flexibility . Very recently, conformational states of metal‐free PsaA in solution have been resolved showing that (a) the C‐terminal lobe is more flexible than the N‐terminal lobe, providing additional proof for the importance of the C‐terminal lobe as recognition site for the permease PsaC, (b) the α‐helical hinge between the two lobes allows a greater conformational flexibility important for ligand binding, and (c) the metal‐binding site is larger and more solvent exposed than indicated by crystallography and in silico simulations . PsaA was shown to bind both manganese and zinc.…”

Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics

“…In order to determine the conformational flexibility of the PsaA protein, five PsaA variants (L56C, S58C, S266C, I125C and I236C) were labelled with the nitroxide MTSL and characterized using cw-EPR. The combination of MD simulations and cw-EPR spectra allowed for elucidation of the flexibility of the PsaA protein lobes, hypothesizing various interactions with other proteins comprising the PsaBCA complex [38]. Since the intrinsic metal (Mn 2 + ) is also paramagnetic, these single variants can be used for distance determination using multior rather high frequency (34 and 94 GHz) cw-EPR as has previous been demonstrated [39].…”

Applications of electron paramagnetic resonance in biomedicine

“…The positions were chosen based on a protein structural analysis (PDBs: 3ZTT [51], 3ZK7 [57], 1PSZ [82]) and MD simulation to access the local protein dynamics [84]. The residues were located on a non-conserved surface and separated by ~20-60 Å to be accessible for DEER measurements.…”

“…For AdcAN RMSF values were calculated using data from the last 250 ns of each of the five independent 750-ns simulations. For PsaA, RMSF values were calculated using data from the final 20 ns of three independent 50-ns simulations from a previous study[84]. In both images the different coloured graphs represent data from independent simulations.…”

“…Backbone atoms of residues 184-194 (flexible helical turn) are shown in the sticks representations. The bars represent the level of distortion in the helical turn as a gradient from blue (no distortion or breakage of hydrogen bonds) to red (distortion or breakage of hydrogen bonds).Deplazes and co-workers performed a further and more detailed MD study of metal-free PsaA in combination with Continuous Wave (CW) EPR of five MTSL-labelled PsaA Cys-mutants[84]. In their work they aimed to build on the study conducted by Counago and co-workers[57] and characterize the flexibility of metal-free PsaA protein in solution, which is not possible from crystallographic data.…”