Binding of single-mutant epidermal growth factor (EGF) ligands alters the stability of the EGF receptor dimer and promotes growth signaling

The epidermal growth factor receptor (EGFR) is a membrane-anchored tyrosine kinase that able to selectively respond multiple extracellular stimuli. Previous studies have indicated the modularity of this system may be caused by ligand-induced differences in stability dimer. However, hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed new approach identify residues responsible for functional divergence selecting (EGF) ligand are conserved among orthologs yet divergent between paralogs. Then, mutated these and assessed mutants' effects on combination molecular dynamics (MD) biochemical techniques. Although EGF mutants had binding affinities EGFR comparable with WT ligand, showed differential patterns phosphorylation cell lines. MD simulations mutations long-range dimer interface. This study shows first time single mutation sufficient alter activation signaling pathway at cellular level. 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Bioinform. 2015; 17: 255-261Crossref (21) Scholar)), included interprotein narrow down those interaction. score calculated sum four components (I: conservation, II: complement paralog's III: coevolution, IV: internal coevolution). Optionally, conducted alignment (multiple [MSTA]) instead sequence (MSA). An implementation done Python. pipeline accepts structure alignments reference conduct analysis. output consists tabular file containing individual scores combined each site, plotted recap S1). algorithm predict 2). Because requires prior knowledge target gene, performed phylogenetic confirm reported monophyletic S2). tree used statistical validation scores. random 53–amino acid–long evolved 100 times Pyvolve (25Spielman Wilke C.O. Pyvolve: flexible Python module simulating sequences along phylogenies.PLoS One. 10e0139047Crossref (41) Assuming distribution will normal, estimated probability without constraints would higher (p-value) (26Chakrabarti Bryant S.H. Panchenko A.R. lies within properties evolutionary changes acids.J. Mol. 2007; 373: 801-810Crossref S3, Table Asn-32, Asp-46, Lys-48, Trp-50 potential candidates divergence. Asn-32 Lys-48 very small both partial (II). Asp-46 relatively (III), S4).Figure 2DIRpred EGF. A, relative ranking outer circle represents (MSA)-based scores, inner (MSTA)-based darker color indicates site-based scoring. values S2. B, cross-conservation plot. plot obtained crossing two Interestingly, no point bottom right half (high ortholog conservation), suggesting independent. observation points out there organism-specific adaptation C, cross-coevolution score. (L-R) (III) y axis ligand–ligand (L-L) (IV) x axis. DIRpred, divergence-inducing prediction; factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Three mentioned previous reports; however, none them individually mutated. tryptophan position 50 strong outlier bioinformatics analysis, Trp-49 Their when do type change into account (data shown). testing outward-facing position, buried contacts (27Gallay Vincent Li de la Sierra I.M. Alvarez Ubieta Madrazo Padron Protein flexibility aggregation factor. time-resolved fluorescence native engineered single-tryptophan mutants.Eur. Biochem. 1993; 211: 213-219Crossref (16) facilitating membrane phospholipids, happens Pro-7 Leu-8 (28Tynan C.J. Roberts S.K. Rolfe Clarke D.T. Loeffler H.H. Kastner Winn M.D. Parker P.J. Martin-Fernandez M.L. Human aligned plasma adopts features Drosophila asymmetry.Mol. Cell 31: 2241-2252Crossref (30) Scholar); burying inside bilayer solution alone (29Li De La Gallay Interaction recombinant phospholipid vesicles. steady-state bis-tryptophan (TRP49-TRP50).Eur. Biophys. 1992; 21: 337-344Crossref (6) although complex example, achieved formation helix, clustering together Val-34 Arg-45 around (30Hommel Harvey T.S. Driscoll P.C. Campbell I.D. High resolution comparison alpha.J. 227: 271-282Crossref (119) Mutation N32R slightly probably due presence guanidinium group R positively charged interact Gln-16 (ECD) S5). corresponding chicken mitogenicity strongly affecting (31Puddicombe Chamberlin S.G. MacGarvie Richter Drummond D.R. Collins Wood Davies significance valine 33 epitope 1996; 271: 15367-15372Abstract (12) literature about before, (32Lahti Lui B.H. Beck S.E. Lee S.S. Ly D.P. Longaker M.T. Yang G.P. Cochran Engineered faster on-rates correlate enhanced activation.FEBS Lett. 585: 1135-1139Crossref (15) Next, manually chose introduce factors. decision given alignment, considering divided groups their kinetics parameters After that, selected infers significant When choices possible, priority ones Freed al designed amino-acid substitution characterization N32R, D46T, K48T, W50Y. To determine integrity secondary substitutions, vitro CD spectroscopy maintained S6). β-sheet content (ranging 0.41 0.54 %) substantially (0.44%), varies. surprising only beta sheet detected CD, (53 acids) β-sheets connected loop regions C N termini flanking. tested ability mutant bind soluble ECD EGFR, isothermal titration calorimetry (ITC) microscale thermophoresis. analogously sample experiments 3). steeper experiments, did appear affect shed light outcome short-term response, sampled expression amount phosphorylated (pEGFR) A431 cells. epidermoid carcinoma overexpresses EGFR. reason, postulated any level amplified. measured total timesteps Western blot (WB) after treating concentrations (100 nM) 4). Remarkably, D46T reduced pEGFR up 30 min treatment 4A). Meanwhile, bands K48T W50Y samples stronger three timesteps. significant. cross-linking assay, follows similar trend 4B). h 6 h, reduction compared control S7). marked samples. inverse relationship quantity, fact explained theory. least ligand. indicate possible alteration recycling observe long-term phenotype, assay IncuCyte live-cell fibroblast lines (Bj5-tα Albino Swiss mouse 3T3). With platform, measuring population, Annexin V green reagent induction apoptosis. day initial incubation, subjected 1, 10, nM concentration Treating Bj5-tα fibroblasts resulted highest proliferation rate control. accompanied morphology signal differentiation (Data decreased suppress 5A). differed most S8A). 3T3 S8B). lines, dependent S9). further apoptosis exhibit treated (>10 (33Gulli L.F. Palmer K.C. Chen Y.Q. Reddy K.B. Epidermal factor-induced reversed reducing activity.Cell Growth Differ. 7: 173-178PubMed decrease population 48 WT, intermediate Cells closest 5B). cells, cell's globular processes increased ratio confluence S10). D46T- K48T-treated signs 5C), S11, Data Significantly, display transduction mechanism. disrupting “loss symmetry” model symmetrical short-lived nature signaling, asymmetric sustained activation, lasting 24 h. Such provides causes modifications behavior experiments. Compared variant constantly lower signal, possibly stable, e