Comparative model building of interleukin-7 using interleukin-4 as a template: A structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation

Using a combination of theoretical sequence structure recognition predictio ns and experimental disulfide bond assignments, a three-dimensional (3D) mo del of human interleukin-7 (hIL-7) was constructed that predicts atypical s urface 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; Waiter MR et al., 1992, J Biol Chem 267:20371-20376). Core secondary struc tures were constructed from sequences of hIL-7 predicted to form helices. T he model was constructed by superimposing IL-7 helices onto the IL-4 templa te and connecting them together in an up-up down-down topology. The model w as finished by incorporating the disulfide bond assignments (Cys3, Cys142), (Cys35, Cys130), and (Cys48, Cys93), which were determined by MALDI mass s pectroscopy and site-directed mutagenesis (Cosenza L. Sweeney E, Murphy JR, 1997, J Biol Chem 272:32995-33000). Quality analysis of the hIL-7 model id entified poor structural features in the carboxyl terminus that, when furth er studied using hydrophobic moment analysis, detected an atypical structur al property in helix D, which contains Cys 130 and Cys142. This analysis de monstrated that helix D had a hydrophobic surface exposed to bulk solvent t hat accounted for the poor quality of the model. but was suggestive of a re gion in IL-7 that maybe important for protein interactions. Alanine (Ala) s ubstitution scanning mutagenesis was performed to test if the predicted aty pical surface chemistry Of. helix D in the hIL-7 model is important for rec eptor activation. This analysis resulted in the construction. purification. and characterization of four hIL-7 variants, hIL-7(K121A), hIL-7(L136A), h IL-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 h IL-7(W143A). which is biologically inactive and displaces [I-125]-hIL-7, is the first reported IL-7R system antagonist.