The Existence of Multiple Conformers of Interleukin-21 Directs Engineering of a Superpotent Analogue

Kent Bondensgaard,Jens Breinholt,Dennis Madsen,Diana Højmark Omkvist,Lishan Kang,Anne Worsaae,Peter Becker,Christine Bruun Schiødt,Siv A Hjorth

doi:10.1074/jbc.m701313200

Kent Bondensgaard, Jens Breinholt + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m701313200

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Abstract

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, alpha-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

Highlights

Within the common ␥ chain family of cytokines, high resolution structural information has been obtained through x-ray crystallography and NMR spectroscopy for IL-2 and IL-4 [3,4,5,6,7,8]
The structure resolved for the fully folded form of human interleukin (hIL)-21 bears a strong resemblance to the structures previously reported for granulocyte-macrophage colony-stimulating factor, IL-2 and IL-4, we have identified some highly distinct features of the hIL-21 molecule
Topology— hIL-21 forms a four-helical bundle arranged in an up-up-down-down topology, which is shared by all members of the family of short-chain cytokines (Fig. 3)

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Met-hIL-21 consists of the sequence of the mature human IL-21 protein as determined by recombinant expression in mammalian cell culture but including an extra methionine residue added recombinantly at the N terminus. The protein is numbered starting with Gln as residue 1. Chemical shift values for HA, CA, CB, N, and CO spectra recorded for hIL-21 display a fairly good dispersion atoms were analyzed to predict ␾ and ␺ backbone angles using despite a number of signals at random coil chemical shift val- the computer program Talos [24]. These latter signals are due to the presence of flexible for 78 residues, and 156 angle ␾/␺ angle constraints were regions in the hIL-21 molecule. The HNHA spectrum, 72 J(HA-HN) scalar coupling constants minor changes were observed in 15N HSQC spectra acquired at were extracted and included in the structure calculations. Different pH values and salt concentrations (pH between 5.0 Hydrogen bond constraints were added for 20 backbone amide and 7.0 and NaCl between 0 and 100 mM). Restricting distance constraints Intraresidue (i ϭ j) Sequential ((i Ϫ j) ϭ 1) Medium range (2 Յ (i Ϫ j) Յ 5) Long range (6 Յ (i Ϫ j))

Dihedral angle constraint violations per Cyana conformer

Stabilization of Helix C and the CD Loop upon Receptor

DISCUSSION