“What I cannot create, I do not understand”
– Richard Feynman
Our research harnesses the power of directed evolution to understand how molecular signals control cellular behavior. Directed evolution enables us to evolve proteins "in a test tube" by generating millions of mutants and then selecting the "winners" with the best functional properties. We study these unique variants to determine how cellular receptors become activated and to guide the development of precisely targeted therapeutics. Our expertise with yeast display allows us to evolve complex eukaryotic proteins, which creates new avenues for the manipulation of difficult-to-target mammalian signaling pathways. Current projects in the lab integrate directed evolution with x-ray crystallography, computational modeling, and cell biology to illuminate receptor systems involved in the development of stem cells.
In vitro characterization
Cellular receptor activation
Protein binding kinetics
Despite the identification of >20,000 genes in the human genome, it is known that only a handful of conserved signaling pathways (Notch, Wnt, Hedgehog, Jak/Stat, PI3K/Akt, TGF-β, NF-κB) control the majority of cell fate decisions during development. However, the related functional properties of these "professional" developmental pathways are carried out by receptors with highly divergent structures and activation mechanisms, which presents unique challenges for the design of therapeutic agonists & antagonists.
We are working to determine how interactions between protein components of developmental pathways transduce signals and in turn specify cell fates. We are especially interested in studying how extracellular posttranslational modifications, such as glycosylation, influence receptor activity. By determining how naturally-occurring modifications regulate signaling, we intend to reverse engineer these systems and develop drugs to mimic their function.
I. Rewiring 'professional' developmental signaling pathways
Notch, Wnt, Hedgehog, and other developmental signaling proteins directly engage receptors & ligands via posttranslational modifications.
Many receptor-ligand pairs form short-lived complexes that are difficult to visualize by traditional methods. This "invisible world" of low-affinity interactions is essential for a myriad of biological functions but is poorly understood due to a near complete absence of structural information. To finally "see" how these proteins engage one another, we are using directed evolution to introduce affinity-enhancing mutations into receptors and ligands, which allows us to capture the complexes for x-ray structure determination. We recently employed affinity-enhanced co-crystallization to determine the first structures of Notch receptor-ligand complexes, and we look forward to applying this method to illuminate other fundamental low-affinity systems.
II. Illuminating the 'invisible world' of low-affinity interactions
III. Targeting Notch: ancient receptor and modern-day oncoprotein
Notch is an evolutionarily ancient receptor that orchestrates the development of all metazoans. In mammals, Notch proteins are master regulators of cell fate decisions, and dysfunctional Notch activity contributes to the pathogenesis of several devastating cancers. Intriguingly, Notch activation may either suppress or promote the growth of tumors depending on cellular context, which necessitates the development of both therapeutic agonists and antagonists. Because global inhibition of Notch is highly toxic, we are developing biologics that can selectively turn Notch receptors "on" or "off" in desired tissues. We recently determined atomic-resolution structures of the Notch1 receptor in complex with its ligands Delta-like 4 (DLL4) and Jagged1 (Jag1), and we are now using these structures to inform the design of synthetic ligands to precisely control Notch activity.
Image courtesy of Eric Smith
Notch-Jagged complex structure juxtaposed with original documentation of the "notched wing" fruit fly phenotype from Sir Thomas Morgan's, "Theory of the Gene" (1917).