Solution nuclear magnetic resonance determination of active site structure for a paramagnetic protein: cyanomet...

The active site structure of a paramagnetic protein, cyanomet myoglobin (Mb) from Aplysia limacina, has been determined in solution by NMR in order to investigate the mechanism of ligand stabilization in this protein, which lacks the usual distal His residue. The structure determination relies on using the experimental dipolar shifts from two-dimensional NMR assignments, NOEs and paramagnetic-induced relaxations as the constraints with a combination of the magnetic susceptibility tensor. The magnetic susceptibility tensor was obtained by using the high resolution crystal co-ordinates of other derivatives of Aplysia Mb (notably metMb, metMbF and metMbN3) for the part of the molecule which is unperturbed by the different ligands, together with the observed dipolar shifts of Aplysia metMbCN, to search for the Euler rotation that correctly converts the crystal co-ordinates to the magnetic axes. The magnetic anisotropies, as well as the orientation of the magnetic axes, are well defined by using the various input data sets based on the conserved structural portions of three sets of crystal co-ordinates. An excellent fit between the calculated versus the observed shifts was obtained for both backbone portions (E, F, G helical segments and FG corner) and the proximal side-chains, which demonstrates that the solution structure of the backbone portions and the proximal side for Aplysia metMbCN is identical to those for three other derivatives investigated by crystallography. On the other hand, the distal side was shown to present minor but important structural changes relative to Aplysia metMbF, which reflect the binding of a diatomic ligand (CN-) rather than a monoatomic one. In particular, the guanidinium group of Arg E10(66) is repositioned and further away from the iron at a distance consistent with H-bonding to the bound cyanide. The bound cyanide is tilted approximately 8 degrees (as detected by the major magnetic axis) away from the heme normal approximately towards the gamma-meso-H. This tilt appears to be due to the attractive hydrogen-bonding with Arg E10(66) and the steric repulsion from Ile E11(67). The results strongly support the role of Arg E10 in stabilizing the anionic ligands by hydrogen bonding as proposed before and demonstrate the validity of the dipolar shifts as a unique and sensitive constraint in determining the active site structure of paramagnetic proteins in the low-spin ferric state.