Solution Dynamics and Secondary Structure of Murine Leukemia Inhibitory Factor: A Four-Helix Cytokine with a Rigid CD Loop

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Abstract
Leukemia inhibitory factor (LIF) is a hematopoietic cytokine which elicits its effects on diverse cell types via both gp130 and a more specific LIF receptor. Recombinant murine LIF was studied by multidimensional homonuclear and 1H−15N heteronuclear NMR and 95% of backbone amide resonances assigned. Definition of the secondary structure by chemical shift data and NOE connectivities shows a four-α-helix bundle fold (helices A−D) in solution, with an additional flexible turn of helix in the AB loop. Subtle differences are seen in the conformations of helices A and D from those defined in the crystal structure [Robinson, R. C., Grey, L. M., Staunton, D., Vankelcom, H., Vernallis, A. B., Moreau, J.-F., Stuart, D. I., Heath, J. K., & Jones, E. Y. (1994) Cell 77, 1101−1116]. The dynamics of the polypeptide backbone of LIF were assessed from 15N T1 and T2 relaxation times and 15N−1H heteronuclear NOEs of the amide groups. Using model-free formalism, the overall rotational correlation time of LIF in solution is calculated to be 9.7 ps. The four α-helices are relatively rigid, and high mobility is observed for N-terminal residues (Ser 1−Asn 21) and the AB loop. In contrast to several closely related cytokines, the long CD loop is relatively rigid. This may have implications for interactions with the specific LIF receptor, which binds in this region.