Asymmetric Usage of Antagonist Charged Residues Drives Interleukin-5 Receptor Recruitment but Is Insufficient for Receptor Activation

Abstract

The cyclic peptide AF17121 (VDECWRIIASHTWFCAEE) is a library-derived antagonist for human Interleukin-5 receptor alpha (IL5Ralpha). We have previously demonstrated that AF17121 mimics Interleukin-5 (IL5) by binding in a region of IL5Ralpha that overlaps the IL5 binding epitope. In the present study, to explore the functional importance of the amino acid residues of AF17121 required for effective binding to, and antagonism of, IL5Ralpha, each charged residue was subjected to site-directed mutagenesis and examined for IL5Ralpha interaction by using a surface plasmon resonance biosensor. One residue, Arg(6), was found to be essential for receptor antagonism; its replacement with either alanine or lysine completely abolished the interaction between AF17121 and IL5Ralpha. Other charged residues play modulatory roles. One class consists of the N-terminal acidic cluster (Asp(2) and Glu(3)) for which alanine replacement decreased the association rate. A second class consists of His(11) and the C-terminal acidic cluster (Glu(17) and Glu(18)) for which alanine replacement increased the dissociation rate. Binding model analysis of the mutants of the latter class of residues indicated the existence of conformational rearrangement during the interaction. On the basis of these results, we propose a model in which Arg(6) and N-terminal acidic residues drive the encounter complex, while Arg(6), His(11), and C-terminal acidic residues are involved in stabilizing the final complex. These data argue that the charged residues of AF17121 are utilized asymmetrically in the pathway of inhibitor-receptor complex formation to deactivate the receptor function. The results also help focus emerging models for the mechanism by which IL5 activates the IL5Ralpha-betac receptor system.

Restricted HIV-1 Env glycan engagement by lectin-reengineered DAVEI protein chimera is sufficient for lytic inactivation of the virus

“…Binding kinetics have not been reported for MVN against HIV-1 gp120 previously. However, interestingly, there is precedent for a protein interaction system in which 1: 1 stoichiometry and conformational change SPR data fits have been observed for the same protein system depending on extent of mutation [35]. …”

“…In contrast, single site binding of MVN that in the current work appears to enable faster on and off rates than observed for CVN, at the same time, allows for greater conformational adaptation in MVN upon gp120 binding. Importantly, more direct biophysical/structural analysis will be required to improve our understanding of the role of conformational changes in MVN and MVN–DAVEI interactions with Env gp120 [35]. …”

Slow-dissociation effect of common signaling subunit β on IL5 and GM-CSF receptor assembly

“…1B). Production and characterization of V5-tagged sIL5Rα were done as previously described [18] [31].…”

Structure-Based Rationale for Interleukin 5 Receptor Antagonism

“…6A), and used SPR technology to directly measure the binding of the mutational variants with IL5R [62] as well as a cell proliferation inhibition assay [63]. We found that modifications of all the charged amino acid residues have an effect on binding of AF17121 to IL5R , and that these residues appear to group into three kinetic classes (Fig.…”

“…In order to seek ideas about structural tendency, we performed molecular dynamics (MD) simulations on AF17121, using the GROMOS96 package of programs and the GROMOS96 43A1 force field [62,64]. Conformation of the major cluster obtained from MD simulation is shown in Fig.…”