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indicates that Cys116 in IL-1Ra is in a homologous position with Phe117 in IL- 1β. The K145D +
Y147T analog lost all detectabled activity (both binding and bioactivity), whereas the Y147G analog
lost all bioactivity but retained 100% binding. These data suggest that Tyr147 is important for
bioactivity of IL-1Ra K145D.
C. Insertion of β Bulge
Page 415
A region of charged amino acids (Gln48 to Asn53, a β bulge) positioned between β strands 4 and 5 has
been implicated in IL-1β binding to its receptor and its immunostimulatory properties [42, 64–67]. The
β-bulge residues form a protrusion on the edge of the open end of the β barrel. Evidence for this patch of
amino acids involved in the function of this molecule comes from mutagenesis studies in which
deletions or substitutions of residues in this region reduced IL- 1β agonist activity without affecting
receptor binding [62,63]. Simoncsits et al. have shown that deletion of amino acids 52–54 (SND) in IL-
1β reduces IL-1RI binding by 10 fold and biological activity by 1000 fold [63]. Also, studies indicate
that a synthetic peptide derived from IL-1β (VQGEESNDK), which contains these six β bulge amino
acids, has immunostimulatory but no inflammatory effects normally associated with IL-1 [64–66]. The
insertion of VQGEESNDK into recombinant human ferritin H chain and recombinant flagellin from
Salmonella muenchen increased the immunogenicity of these antigens in mice [67].
Greenfeder et al. inserted this β-bulge region into IL-1Ra K145D either after Ile51 of after Pro53 [61].
The insertion of the β bulge (QGEESN) after either position 51 or 53 of IL-1Ra K145D resulted in
analogs that retained full IL-1RI binding and increased bioactivity by 3–4 fold. Aslo they tried to obtain
the analogs of the QGEESN insertion in the absence of the K145D mutation either after amino acid 51
or 53 of IL-1Ra. None of the plasmid clones with the insertion at position 51 of IL-1Ra produced the
appropriate protein, whereas they were able to isolate clones with the insertion at position 53. Based on
these results, Greenfeder et al. suggest that in the first case the insertion interfered in the proper folding
of the protein, whereas the second mutant folded properly and exhibited only 10 to 20% of the IL-1RI
binding activity. The mutants with K145D + QGEESN insertion after Ile51 of IL-1Ra showed an
increase in bioactivity in the range of 3–8 fold. The triple mutant IL-1Ra K145D/H54P/QGEESN
showed a higher bioactivity, and based on their mutagenesis study, Greenfeder et al. suggest that this
increase in activity may be due to the introduction of Pro and not the removal of His at position 54 in the
K145D mutant of IL-1Ra. The cumulative effects of these three mutations are also interesting since their
positions on the IL-1Ra protein appear to be spatially separated. The residues Ile51 and His54 are
located on the open face of the β barrel of IL-1Ra, whereas Lys145 is located away from the open barrel
end. In IL-1β, the same relative
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