Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization

Benjamin Bardiaux^1,6, Adrien Favier², Manuel Etzkorn⁴, Marc Baldus⁵, Anja Böckmann³, Michael Nilges¹, Thérèse E Malliavin¹

¹Unité de Bioinformatique Structurale, CNRS URA 2185, Institut Pasteur, Paris, France; ²Institut de Biologie Structurale Jean-Pierre Ebel CEACNRS-Université Joseph Fourier, Grenoble, France; ³Institut de Biologie et Chimie des Proteines, UMR 5086 CNRS/Université de Lyon 1, Lyon, France; ⁴Max Planck Institute Biophysical Chemistry, Göttingen, Germany; ⁵Bijvoet Center for Biomolecular Research Faculty of Science, Utrecht University Utrecht, The Netherlands; ⁶Campus Berlin-Buch, Berlin, Germany

Abstract: We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context.

Keywords: structural bioinformatics, NMR structure calculation, ARIA, non-crystallographic symmetry, crystallographic ensemble refinement, molecular dynamics simulation