Using a combination of theoretical sequence structure
recognition predictions and experimental disulfide bond
assignments, a three-dimensional (3D) model of human interleukin-7
(hIL-7) was constructed that predicts atypical surface
chemistry in helix D that is important for receptor activation.
A 3D model of hIL-7 was built using the X-ray crystal structure
of interleukin-4 (IL-4) as a template (Walter MR et al.,
1992, J Mol Biol. 224:1075–1085; Walter
MR et al., 1992, J Biol Chem 267:20371–20376).
Core secondary structures were constructed from sequences
of hIL-7 predicted to form helices. The model was constructed
by superimposing IL-7 helices onto the IL-4 template and
connecting them together in an up–up down–down
topology. The model was finished by incorporating the disulfide
bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48,
Cys93), which were determined by MALDI mass spectroscopy
and site-directed mutagenesis (Cosenza L, Sweeney E, Murphy
JR, 1997, J Biol Chem 272:32995–33000).
Quality analysis of the hIL-7 model identified poor structural
features in the carboxyl terminus that, when further studied
using hydrophobic moment analysis, detected an atypical
structural property in helix D, which contains Cys130 and
Cys142. This analysis demonstrated that helix D had a hydrophobic
surface exposed to bulk solvent that accounted for the
poor quality of the model, but was suggestive of a region
in IL-7 that maybe important for protein interactions.
Alanine (Ala) substitution scanning mutagenesis was performed
to test if the predicted atypical surface chemistry of
helix D in the hIL-7 model is important for receptor activation.
This analysis resulted in the construction, purification,
and characterization of four hIL-7 variants, hIL-7(K121A),
hIL-7(L136A), hIL-7(K140A), and hIL-7(W143A), that displayed
reduced or abrogated ability to stimulate a murine IL-7
dependent pre-B cell proliferation. The mutant hIL-7(W143A),
which is biologically inactive and displaces
[125I]-hIL-7, is the first reported IL-7R