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


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.

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    • "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.] "
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    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.
    Protein Science 07/2015; DOI:10.1002/pro.2755 · 2.85 Impact Factor
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    • "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 Figure 6. "
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    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.
    Cell 04/2015; 161(3):634-646. DOI:10.1016/j.cell.2015.03.006 · 32.24 Impact Factor
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    • "Previous studies with EGF mutants have revealed that some amino acid residues in EGF (Tyr-13, Leu-15, His-16, Arg-41, Gln-43 and Lue-47)3738 together with Gly-1239 play a key role for EGFR binding and/or mitogenicity in mammalian cells (Supplementary Table S3). Furthermore, by using a fluorescence-labeled EGFR extracellular domain as a probe for EGF mutants displayed on the yeast cell surface, Cochran et al.40 and Lahti et al.41 have identified and analyzed various EGFR agonists, in which mutant 114, 28 and 123 possess over 30-fold affinity to EGFR compared with wild-type EGF4041. When comparing the amino acid sequences among the previously reported EGF mutants and the HLH peptides harboring EGFR agonistic activity (see Supplementary Table S3), the HLH peptides are fundamentally different from these EGF mutants not only in primary structure but also secondary structure. "
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    ABSTRACT: Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.
    Scientific Reports 02/2014; 4:4242. DOI:10.1038/srep04242 · 5.58 Impact Factor
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