Determination of the relative orientation of the two halves of the domain-swapped dimer of cyanovirin-N in solution using dipolar couplings and rigidbody minimization

The HIV-inactivating protein cyanovirin-N (CVN) exists in two forms that ar e pH- and solvent-dependent: a monomer which predominates at neutral pH (gr eater than or equal to 90%) and a symmetric domain-swapped dimer. We have i nvestigated the orientation of the two halves of the domain-swapped dimer o f CVN at neutral pH in solution using dipolar couplings measured in a neutr al liquid crystalline bicelle medium. D-1(NH) dipolar couplings for the dim er were readily measured for 18 out of 101 residues, and are shown to be in consistent with the orientation of the two halves of the dimer observed in the X-ray structure obtained from crystals grown at low pH in the presence of organic solvent. The orientation of the two halves of the domain-swapped dimer was determined by rigid body minimization, subject to the requiremen ts of C-2 symmetry. The starting coordinates for the calculations consisted of the X-ray coordinates for the two halves (with the linker residues dele ted), separated by similar to 45 Angstrom and placed in three different rel ative orientations. One-half of the dimer is held fixed, the other half is free to rotate and translate (6 degrees of freedom), and the alignment tens or for the dipolar couplings is free to rotate (3 degrees of freedom). The target function comprised only four terms: dipolar coupling restraints (18 x 2), distance restraints (12) to link the two halves and to prevent steric clash, a radius of gyration restraint to achieve appropriate compaction, a nd a quartic van der Waals repulsion term. Structures were calculated for d ifferent target values of the radius of gyration, and back-calculation of t he alignment tensor and dipolar couplings on the basis of molecular shape w as used to filter the resulting structures, Prediction of dipolar couplings in this manner is predicated on the assumption that orientational order is dictated by steric interactions between the liquid crystalline medium and the protein. The validity of this assumption in this particular case is evi denced by the excellent agreement between predicted and observed dipolar co uplings for the monomer. We show that the data is only consistent with a ve ry small range of orientations of the two halves of the dimer in which the angle between the long axes of the two halves is similar to 110 degrees. Th e relative orientation of the two halves of the dimer at neutral pH in solu tion is quite different from that observed in the crystals obtained at low pH in organic solvent. The factors stabilizing the relative orientation of the two halves of the dimer under different conditions are discussed. The m ethodology presented in this paper should find a wide range of applicabilit y to numerous other structural problems involving multimeric proteins and p rotein-protein complexes.