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

Abstract

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 @ 125 I#-hIL-7, is the first reported IL-7R system antagonist.

Structural and Biophysical Studies of the Human IL-7/IL-7Rα Complex

“…The disulfide bonds are between C34-C129, C47-C141, and C2-C92. This disulfide bond patterning is in contrast to homology models of IL-7 and biochemical data that predicted different locations by swapping the C47-C141 and C2-C92 disulfide bonds for C2-C141 and C92-C141 (Cosenza et al, 2000; Cosenza et al, 1997). While the electron density allowed tracing of the C34-C129 and C47-C141 disulfide bonds, it was too weak near the N-terminus (residues 1-6(7)) and the end of helix C proceeding into the second crossover loop (residues 89-123) to trace the third disulfide bond between C2-C92.…”

“…Previous studies indicated that W142 of helix D, the sole Trp of IL-7, was critical to the interaction between IL-7 and γ c (Cosenza et al, 2000; vanderSpek et al, 2002). However, the IL-7 structures show the burial of W142 into the hydrophobic core of the 4-helix bundle with the Nε forming a hydrogen bond (H-bond) to T86 Oγ1 (Figure 2B).…”

A combinatorial approach of N-terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL-7 in E. coli fed-batch culture

“…Moreover, this strategy is expected to result in an authentic N-terminus of the fusion partner due to the high sequence specificity of SUMO protease (Butt et al 2005). The conformational property of purified recombinant hIL-7 from CD spectroscopy showed a typical helical structure with 31.5 % α-helix and 26.43 % β-sheet, which agreed well with the established literature report (Cosenza et al 2000). The laboratory-produced recombinant hIL-7 supported 2E8 cell growth at a protein concentration of as low as 1 ng/mL, which was also a recommended concentration for the commercial standard.…”

Murine and Human IL-7 Activate STAT5 and Induce Proliferation of Normal Human Pro-B Cells

“…We were therefore surprised to discover that murine IL-7 can transduce signals through the IL-7R on human B-lineage cells. A previously published study offers a possible explanation (48). A virtual construction of a three-dimensional model of IL-7 structure was created, and a sequence bounded by Cys 131 and Cys 143 containing a potential hydrophobic surface was hypothesized to be important for protein interactions.…”