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

Abstract

The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using ¹⁵N 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 ¹⁵N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D_∥/D_⊥ of 1.31 and an effective correlation time (4D_⊥ + 2D_∥)⁻¹ of 8.9 ns. The average values of the order parameters (S²) for the four helices, 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-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S² values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S² values, implying higher amplitude motions than for the backbone of sites I and II. 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 months in ²H₂O at 37 °C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.