H-1, C-13, AND N-15 NMR RESONANCE ASSIGNMENTS, SECONDARY STRUCTURE, AND BACKBONE TOPOLOGY OF A VARIANT OF HUMAN INTERLEUKIN-3

Interleukin-3 (IL-3) is a cytokine which stimulates the proliferation and differentiation of hematopoietic progenitors into multiple cell li neages. The H-1, N-15, and C-13 NMR resonances of a recombinant human IL-3 variant (SC-65369) have been assigned using two- and three-dimens ional NMR techniques on uniformly C-13/N-15-enriched protein. Five hel ical segments (residues 16-26, 42-50, 55-65, 73-82, and 104-120) and t hree reverse turns (residues 51-54, 68-71, and 87-90) were identified from the pattern of sequential NOE connectivities, NH(i)-(CH)-H-alpha( i) scalar coupling constants ((3)J(NH alpha)), amide hydrogen exchange data, and the deviation of C-13(alpha), C-13(beta), (CO)-C-13, and (C H)-H-alpha chemical shifts from random-coil values. Long-range NOEs in dicate that the global folding pattern of human IL-3 is a four-helical bundle with an up-up-down-down arrangement of helices that is similar to that of other members of the cytokine family, such as granulocyte- macrophage colony stimulating factor (GM-CSF). A fifth short helix (he lix A', residues 42-50) is located in the loop connecting the first an d second helices. The absence of helix A' in the corresponding structu res of GM-CSF and interleukin-5 suggests that it may be important for recognition of IL-3 by its receptor. The existence of at least two for ms of the protein that differ in local conformation was implied from t he observation of a limited set of doubled resonances in which each do ubler partner had a similar pattern of short-, medium-, and long-range NOEs. The majority of the doubled resonances were close in sequence o r space to a proline-rich sequence, which suggested that the different conformational forms of SC-65369 may be caused by slow cis-trans isom erization of proline peptide bonds.