Cochran Lab
Cochran Lab
RESEARCH

Engineering Growth Factor Ligand and Receptor Interactions

Introduction

Our research group has much interest in engineering natural growth factor ligands and receptors as molecular tools for studying the relationship of protein sequence/structure to biological read-outs. These engineered proteins also have important clinical applications as therapeutics for the treatment of a variety of human malignancies.

Figure 1
Fig. 1. Growth factor efficacy is dictated by many factors, including ligand/receptor binding affinity, and cell internalization and trafficking.

Receptor binding interactions are only one of many factors that determine a ligand’s biological effect. After a ligand binds to a cell surface receptor, other processes, such as internalization, recycling, and degradation, can directly affect which signal pathways are activated and the strength and lifetime of the signal (Figure 1). Ligand-receptor binding affinity, kinetics and the pH dependence of binding also impact these biological processes. We engineer proteins with altered biochemical and biophysical properties to study and define these relationships for receptor agonists or antagonists, and to elucidate the molecular mechanisms of receptor-mediated signal transduction. We also work with physician-scientists and commercial partners to translate these engineered proteins into the clinic.

For an overview of this approach, please see our review by Jones et al., entitled “Developing Therapeutic Proteins by Engineering Ligand-Receptor Interactions” (Trends in Biotechnology, 26, 498-505, 2008).

 

Examples of on-going projects include:

Project 1

Figure 2Fig. 2. Structural model of EGF in complex with EGFR. Modified from PDB 1IVO.

Engineering a toolkit of epidermal growth factor (EGF) agonists: We engineered a toolkit of EGF ligands to study the relationship between biochemical properties and biological efficacy, and to identify enhanced EGF agonists for wound healing applications. The EGF receptor (EGFR) is a transmembrane receptor tyrosine kinase that regulates a myriad of biological process through a complex network of signaling pathways. The EGF/EGFR system has been extensively studied to understand fundamental biological processes, including signal transduction mechanisms and intracellular receptor trafficking. Until now, the majority of experimental and computational studies have focused on the effects of a few endogenous ligand/receptor combinations with discrete biochemical profiles; thus, it has not been possible to rank the relative importance of binding affinity, kinetic binding rates, and pH-dependent binding on biological read-outs. We characterized the biochemical properties of more than 40 engineered EGF ligands, discovered through our combinatorial protein engineering efforts, and identified 5 classes of ligands with different affinities and kinetic rates of EGFR binding: 1) ligands with similar off-rates but different on-rates, 2) ligands with similar on-rates but different off-rates, 3) ligands with similar affinities at extracellular (physiological) pH 7.5, but different affinities at endosomal pH 5.5, 4) ligands with similar affinities at pH 5.5, but different affinities at pH 7.5, and 5) ligands with similar off-rates at pH 7.5 and similar affinities at pH 5.5, but different on-rates at pH 7.5. We are using these EGF mutants to correlate biochemical/biophysical properties with biological activity. For example, we showed that EGF mutants with faster EGFR binding on-rates elicited enhanced EGFR activation and EGFR downregulation compared to wild-type EGF (Lahti et al., 2011 FEBS Letters 585, 1135-1139). In addition, based on knowledge gained from our studies, we rationally engineered several EGF mutants with altered biochemical profiles. In addition to increased EGFR binding on-rates, these mutants have increased kinetic off-rates and display pH-dependent EGFR binding. The latter two properties should result in a smaller ligand-occupied fraction of internalized receptors, potentially leading to increased receptor recycling and sustained signaling. We are currently testing if these EGF mutants elicit enhanced biological activity in fibroblasts compared to wild-type EGF.

In an alternative strategy, in collaboration with James Swartz (Stanford Chemical Engineering), we developed a cell-free protein expression platform for producing mutant EGF libraries in microtiter plates and screening them based on biological read-outs, in contrast to most protein engineering methods which rely on screens based on binding affinity (Lui, et al., 2011 J. Mol. Biol. 413, 406-415). As a result, we identified a first-in-class EGF mutant that stimulates significantly enhanced fibroblast proliferation compared to wild-type EGF. We are currently characterizing the biochemical properties of this engineered EGF mutant and its effects on EGFR binding, activation, and cell trafficking.

Figure 3Fig. 3. Diagram of our platform’s six steps to screen a library of protein mutants for improved biological activity. From Lui et al., J. Mol. Biol. 2011.

 

Project 2

Figure 4Fig. 4. Structure of NK1 homodimer indicating location of our mutations. Models were generated using Pymol and PDB 1NK1. From Jones et al., Proc. Nat. Acad. Sci. 2011.

Engineering agonists and antagonists based on hepatocyte growth factor (HGF): The Met receptor tyrosine kinase and its ligand HGF play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally-occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting biophysical studies and its therapeutic potential. We engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists (Jones et al., 2011 Proc. Nat. Acad. Sci. 108, 13035-40). We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. Interestingly, NK1 variants isolated from our library screens functioned as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. Next, we introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally-engineered antagonists exhibited melting temperatures up to ~64 °C, a 15 °C improvement over antagonists derived from wild-type NK1, and ~40-fold improvement in recombinant expression yield in yeast. We are currently parsing the contribution of individual mutations to stability, expression, and Met binding. To create potent agonists, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. Remarkably, these covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, suggesting that Met activation is mediated through a receptor clustering mechanism rather than through allosteric changes. These engineered NK1 agonists will open up new research areas where HGF has shown great promise in liver, renal, and cardiac tissue engineering, but has been limited due to HGF instability or the inability to generate adequate amounts of recombinant protein. In addition, we are using these NK1 mutants as tools to further elucidate molecular determinants of Met receptor activation.