Mutant proteins of human interleukin 2

Abstract

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (112) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein.The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined.All muteins besides two (K44/T125 and QIlO/T125) could be renatured and purified. One mutein (K94/T12.5) exhibited a more than tenfold-improved renaturation yield.One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and highaffinity receptor binding. The binding to the low-affinity 112-binding protein (Tac antigen) was unimpaired.A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high-and the low-affinity receptor, however, was reduced about 20-fold.Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins.It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.The transient synthesis of interleukin 2 (112) and its receptor by activated T lymphocytes apparently represents an essential step in the elaboration of the cellular immune response (for recent reviews see [I -31). The binding of 112 to a highaffinity receptor (& = 10 pM) provides the growth-signal leading to the clonal expansion of antigen-specific T cells.Interestingly, the high-affinity receptor has been established to be assembled from a low-affinity (Kd z 10 nM) 112- large extent preliminary, however, since the proteins were not renatured and purified before measuring proliferative or receptor-binding activity. Thus, effects of a certain mutation on the folding or on the receptor-binding sites could not be clearly differentiated.During the studies presented in this paper a series of charged residue positions and of hydrophobic positions were exchanged which cover more or less the whole surface of 112. The muteins were all submitted to a renaturation and purification protocol before analysing biological activities. Two muteins appeared which define positions involved in high-or in low-affinity receptor binding. Surprisingly, one mutein was obtained exhibiting a large increase in yield during renaturation. METHODS Construction of mutant strainsA cDNA coding for the mature part of human I12 [8] plus an initiator methionine [I91 was submitted to oligonucleotidedirected mutagenesis by the gapped duplex DNA approach [20, 211. The synthetic oligonucleotides comprised 6-16 residues before and after the nucleotide(s) to be exchanged, and they were provided by Dr H. Blocker (GBF, Braunschweig) or synthesised on a DNA synthesiser (Applied Biosystems, model 380). Mutation...

Efficacy of interspinous process lumbar fusion with recombinant human bone morphogenetic protein-2 delivered with a synthetic polymer and β-tricalcium phosphate in a rabbit model

“…Dimeric E-BMP-2 was purified using several steps of chromatography. Details of the procedures for dimerization of cytokines have already been reported [12,32,42,43].…”

Binding of small molecules to an adaptive protein–protein interface

“…2a). Previous mutational work (17)(18)(19) has identified residues important for binding of IL-2 to the IL-2 receptor (a ''hot spot''). Even though Compound 1 was not designed to bind IL-2, it does bind directly over the region of IL-2 that is functionally critical for binding to IL-2R␣ (Fig.…”

“…None of the cysteine substitutions caused a substantial change in the ability of IL-2 to bind to IL-2R␤ (Table 2), suggesting they did not significantly perturb the conformation of the protein. Some of the mutants (Y45C, L72C, and K43C) did interfere with IL-2R␣ binding, as anticipated from previous mutagenesis studies that define the IL-2 hot spot (17)(18)(19). Each cysteine variant was screened against a 7,000-compound tethering library (6) in pools of 10 compounds each.…”

Receptor binding properties of four‐helix‐bundle growth factors deduced from electrostatic analysis

“…The spatial conservation of both the cytokines and their receptors suggests that the 3D structure of their complexes may also be conserved. Immunochemical experiments and experiments on recombinant analogs of murine (Zurawski & Zurawski, 1989, 1992Zurawski et al, 1990) and human IL-2 (Liang et al, 1986;Weigel et al, 1989;Landgraf et al, 1992), IL-4 (Kruse et al, 1991(Kruse et al, , 1992(Kruse et al, , 1993Ramanathan et al, 1993), and CM-CSF (Kaushansky et al, 1989;Lopez et al, 1992;Meropol et al, 1992) suggest that all of them are likely to interact with their receptors in an orientation similar to hGH (de Vos et al, 1992), i.e., with the surface involving the A, C, and D helices. Mapping of functionally important residues onto the crystal structures of IL-2 and IL-4 indicates that a region near the C-terminus is one of the potential receptor binding sites.…”

“…There is little cross-reactivity between hormones and receptors of different species or different types, unless (1) both are highly homologous (Fukunaga et al, 1991;Nicola & Metcalf, 1991), or (2) the hormones share a common receptor component (Boulay & Paul, 1992;Kondo et al, 1993;Russell et al, 1993), or (3) have different binding sites on the hormone (Fuh et al, 1993). An indication that a part of the specificity of hormone-receptor recognition may be dependent on electrostatic interactions is given by observations that mutations involving charged residues in 1L-2 (Weigel et al, 1989;Zurawski et al, 1990;Zurawski & Zurawski, 1992), IL-4 (Kruse et al, 1992Ramanathan et al, 1993;Wlodaweret al, 1993), GM-CSF(Kaushanskyetal., 1989;Lopez et al, 1992), and hGH (Cunningham et al, 1991;Cunningham & Wells, 1993;Wells & de Vos, 1993) and its receptor (Bass et al, 1991) often have a significant effect on receptor binding. We have therefore carried out a comparative study of the electrostatic properties of 5 growth factors, the extracellular part of the hGH receptor, and a homology-modeled extracellular part of the 130-kDa chain (IL-4R) of the IL-4 receptor in order to examine their binding modes.…”