Secondary structure and topology of human interleukin 4 in solution

Abstract

Human interleukin 4 (IL-4) has been studied by 2D and 3D NMR techniques using uniformly 15N-labeled recombinant protein. Assignment of resonances for all but 3 of the 130 residues of the recombinant protein has been achieved, enabling the secondary structure of the protein to be defined. This consists of four major alpha-helical regions and one short section of double-stranded antiparallel beta-sheet. Analysis of distance and angle restraints derived from NMR experiments has enabled the overall molecular topology to be determined. This is related to that found for other four-helix proteins but has several distinctive features including cross-linking of helices by means of three disulfide bonds and a short section of beta-sheet. The structural analysis gives support to the hypothesis that many helical cytokines have a common fold and provides a basis for understanding the biological function of IL-4.

Aspects of Receptor Binding and Signalling of Interleukin-4 Investigated by Site-directed Mutagenesis and NMR Spectroscopy

“…Approximately 60 NOE of the Na:N-and aNN-type (Oschkinat et al, 1990) were used for the sequential assignment, supplementing the data from the heteronuclear spectra. The results obtained werein line with the assignments reported in the Iiterature (Redfield et al, 1991;Powers et al, l992b) except that the assignment of the methyl group proton resonances of A49 was very differenttothat obtained by Powers et al In our spectra, the proton methyl signal occurs at a chemical shift of 0·28 p.p.m. It was assigned by means ofCOSY, TOCSY and NOESY cross peaks, and showed an unexpectedly large line width.…”

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy

“…Further, the packing of the helices is different. In particular, the preliminary Redfield et al (1991) structures have a more open conformation than that Interleukin-4 Structure and Folding depicted in Figure 8 and implied by the NOE data in Figure 7. The reason for this difference can be attributed to the fact that all interhelical NOEs involve side chains.…”

“…Comparison of the Secondary Structure with That Determined by Redfield et al (1991). In a recent paper, Redfield et al (1991) presented the lSN and 'H backbone assignments of IL-4 together with a secondary structure determination based on 3D lSN-edited NOESY spectroscopy.…”

“…The reason for this difference can be attributed to the fact that all interhelical NOEs involve side chains. As Redfield et al (1991) did not have 13C-labeled IL-4 at their disposal, they were only able to obtain a very limited number of 'H side-chain assignments and had to rely on conventional 2D 'H-lH NOESY spectroscopy to identify these NOEs. Consequently, they could not observe interhelical NOEs between as many residue pairs as in the current work and were unable to observe any NOEs between helices A and B and between helices C and D. In contrast, we were able to identify a much larger network of interhelical NOEs involving all pairs of helices from the 3D 13C-edited NOESY spectrum which place much more stringent restrictions on the helix packing (cf.…”

Three-dimensional Solution Structure and Backbone Dynamics of a Variant of Human Interleukin-3

“…The sufficiency levels of the models are similar to those observed for other globular proteins (cf. Redfield et al, 1992;Farrow et al, 1994). The isotropic motion assumption in the model-free formalism thus appears to be valid, even though the three principal components of the inertia tensor of SC-65369 have a ratio of 1.00:0.64:0.40.…”