Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine

The backbone dynamics of the four-helical bundle cytokine leukemia inhibito ry factor (LIF) have been investigated using N-15 NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by N-15 re laxation measurements, the dynamics parameters were calculated using the mo del-free formalism incorporating the model selection approach. The principa l components of the inertia tensor of MH35-LIF as calculated from its NMR s tructure, were 1:0.98:0.38. The global rotational motion of the molecule wa s, therefore, assumed to be axially symmetric in the analysis of its relaxa tion data. This yielded a diffusion anisotropy D-\\/D-perpendicular to of 1 .31 and an effective correlation time (4D(perpendicular to) + 2D(\\))(-1) o f 8.9 ns. The average values of the order parameters (S-2) for the four hel ices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-L IF were higher than those of their counterparts in short-chain members of t he four-helical bundle cytokine family. Residues involved in LIF receptor b inding showed no consistent pattern of backbone mobilities, with SZ values ranging from 0.71 to 0.95, but residues contributing to receptor binding si te III had relatively lower S2 values, implying higher amplitude motions th an for the backbone of sites I and Il. In the relatively slow motion regime , backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several month s in (H2O)-H-2 at 37 degrees C. Evidence for local unfolding was considered , and correlations among various structure-related parameters and the backb one amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.