Article

Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation

Department of Bioengineering, Cancer Center, Bio-X Program, Stanford University, Stanford, CA 94305, USA.
FEBS letters (Impact Factor: 3.17). 03/2011; 585(8):1135-9. DOI: 10.1016/j.febslet.2011.03.044
Source: PubMed

ABSTRACT

Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.

Full-text preview

Available from: ncbi.nlm.nih.gov
    • "Yeast-surface displayed libraries of EGF mutants, created by error-prone PCR and DNA shuffling, were screened to identify variants with up to a 30-fold increase in binding to the extracellular domain of EGFR[21]. Two EGF variants were found to possess faster EGFR binding on-rates and were more potent at stimulating EGFR activation[49]. In a different study, rationally created chimeric proteins consisting of EGF and a related mitogen TGFa were shown to be active at 10-fold lower concentrations than their wild-type ligand counterparts; these variants were shown to possess 3-to 5-fold higher binding on-rates and off-rates compared to natural EGFR ligands[50]. These results demonstrate that improved potency can be achieved by engineering growth factor variants with altered kinetic binding parameters. "
    [Show abstract] [Hide abstract] ABSTRACT: Statement of significance: Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.
    No preview · Article · Nov 2015 · Acta biomaterialia
  • Source
    • "Notably, it was shown that 60% of protein-small molecule, 90% of protein-protein, and 85% of protein-nucleic acid complexes have at least one titratable residue that changes its protonation state upon binding at physiological pH (6.5) [1]. [Although many chemical and biological binding reactions are pH-dependent [2] [3] [4], the effect of pH has been generally neglected for binding free energy calculations because there were hardly any methods to accurately model the effect.] "
    [Show abstract] [Hide abstract] ABSTRACT: We present a computational scheme to compute the pH-dependence of binding free energy with explicit solvent. [Despite the importance of pH, the effect of pH has been generally neglected in binding free energy calculations because of a lack of accurate methods to model it.] To address this limitation, we use a constant-pH methodology to obtain a true ensemble of multiple protonation states of a titratable system at a given pH and analyze the ensemble using the Bennett acceptance ratio (BAR) method. The constant pH method is based on the combination of enveloping distribution sampling (EDS) with the Hamiltonian replica exchange method (HREM), which yields an accurate semi-grand canonical ensemble of a titratable system. By considering the free energy change of constraining multiple protonation states to a single state or releasing a single protonation state to multiple states, the pH dependent binding free energy profile can be obtained. We perform benchmark simulations of a host-guest system: cucurbit[7]uril (CB[7]) and benzimidazole (BZ). BZ experiences a large pKa shift upon complex formation. The pH-dependent binding free energy profiles of the benchmark system are obtained with three different long-range interaction calculation schemes: a cutoff, the particle mesh Ewald (PME), and the isotropic periodic sum (IPS) method. Our scheme captures the pH-dependent behavior of binding free energy successfully. Absolute binding free energy values obtained with the PME and IPS methods are consistent, while cutoff method results are off by 2 kcal/mol. We also discuss the characteristics of three long-range interaction calculation methods for constant-pH simulations. This article is protected by copyright. All rights reserved. © 2015 The Protein Society.
    Preview · Article · Jul 2015 · Protein Science
  • Source
    • "The striking decrease in K d is driven by a large increase in the association rate constant (k on ) with no apparent change in dissociation kinetics (k off ). While relatively unusual (Anderson et al., 1998 ), a small number of protein-receptor affinity mutations are known to specifically affect k on (Lahti et al., 2011; Lengyel et al., 2007). Our findings strongly suggest that RBP-STRA6 docking involves a conformational adaptation of RBP and that this initial step, rather than diffusion, limits the binding reaction, consistent with a selected-fit model (Weikl and von Deuster, 2009). "
    [Show abstract] [Hide abstract] ABSTRACT: Gestational vitamin A (retinol) deficiency poses a risk for ocular birth defects and blindness. We identified missense mutations in RBP4, encoding serum retinol binding protein, in three families with eye malformations of differing severity, including bilateral anophthalmia. The mutant phenotypes exhibit dominant inheritance, but incomplete penetrance. Maternal transmission significantly increases the probability of phenotypic expression. RBP normally delivers retinol from hepatic stores to peripheral tissues, including the placenta and fetal eye. The disease mutations greatly reduce retinol binding to RBP, yet paradoxically increase the affinity of RBP for its cell surface receptor, STRA6. By occupying STRA6 nonproductively, the dominant-negative proteins disrupt vitamin A delivery from wild-type proteins within the fetus, but also, in the case of maternal transmission, at the placenta. These findings establish a previously uncharacterized mode of maternal inheritance, distinct from imprinting and oocyte-derived mRNA, and define a group of hereditary disorders plausibly modulated by dietary vitamin A. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Cell
Show more