Differential Isotope Labeling Strategy for Determining the Structure of Myristoylated Recoverin by NMR Spectroscopy

The three-dimensional solution structure of recombinant bovine myristoylated recoverin in the Ca2+-free state has been refined using an array of isotope-assisted multidimensional heteronuclear NMR techniques. In some experiments, the myristoyl group covalently attached to the protein N-terminus was labeled with 13C and the protein was unlabeled or vice versa; in others, both were 13C-labeled. This differential labeling strategy was essential for structural refinement and can be applied to other acylated proteins. Stereospecific assignments of 41 pairs of β-methylene protons and 48 methyl groups of valine and leucine were included in the structure refinement. The refined structure was constructed using a total of 3679 experimental NMR restraints, comprising 3242 approximate interproton distance restraints (including 153 between the myristoyl group and the polypeptide), 140 distance restraints for 70 backbone hydrogen bonds, and 297 torsion angle restraints. The atomic rms deviations about the averaged minimized coordinate positions for the secondary structure region of the N-terminal and C-terminal domains are 0.44 ± 0.07 and 0.55 ± 0.18 Å for backbone atoms, and 1.09 ± 0.07 and 1.10 ± 0.15 Å for all heavy atoms, respectively. The refined structure allows for a detailed analysis of the myristoyl binding pocket. The myristoyl group is in a slightly bent conformation: the average distance between C1 and C14 atoms of the myristoyl group is 14.6 Å. Hydrophobic residues Leu28, Trp31, and Tyr32 form a cluster that interacts with the front end of the myristoyl group (C1-C8), whereas residues Phe49, Phe56, Tyr86, Val87, and Leu90 interact with the tail end (C9-C14). The relatively deep hydrophobic pocket that binds the myristoyl group (C14:0) could also accommodate other naturally occurring acyl groups such as C12:0, C14:1, and C14:2 chains.