# Illuminating Notch receptor-ligand selectivity through structure-guided protein engineering

> **NIH NIH R35** · H. LEE MOFFITT CANCER CTR & RES INST · 2021 · $430,000

## Abstract

PROJECT SUMMARY
Overall vision of the research program. My research is focused on understanding the “molecular language”
of cell-to-cell communication. Cell surface receptors transmit signals in response to external cues, and receptor
activity is regulated by extrinsic factors such as posttranslational modifications, mechanical force, and chemical
environment. Using a multidisciplinary approach that leverages my training in structural biology, biophysics, and
protein engineering, my group is attempting to solve difficult problems in the area of receptor biology. We
hypothesize that the distinct structural and biophysical parameters governing receptor-ligand interactions control
downstream signaling, and that ligands may be engineered to alter these parameters and reprogram signaling
outputs. My long-term goal is to harness the insight obtained through our mechanistic studies to design proteins
that can fine-tune receptor activity and function as powerful next-generation therapeutics.
Overview of research in the laboratory. Notch signaling is initiated when a mechanosensory Notch receptor
is “pulled on” by a Delta-like (DLL) or Jagged (Jag) ligand expressed on an adjacent cell. In mammals, the
various Notch receptor (Notch1-4) and ligand (DLL1, DLL4, Jag1, Jag2) subtypes are able to induce distinct or
even opposing cellular responses. Ligand selectivity is further regulated by Notch receptor glycosylation, which
inhibits the activity of Jag ligands yet potentiates the activity of DLL ligands. We currently have a poor
understanding of the molecular basis for ligand discrimination in the Notch pathway, and structural studies of
Notch receptor-ligand interactions have been difficult because their nearly undetectable binding affinity precludes
reconstitution of stable complexes. We recently overcame this obstacle by using directed evolution to strengthen
DLL4 and Jag1 binding to Notch1, which allowed us to capture their interacting domains for x-ray crystal structure
determination. My lab is now building upon these transformative results to explore how the structural, biophysical,
and mechanical properties of various ligand subtypes contribute to their unique functions.
Goals for the next five years. Over the five-year funding period, the goals of this project are to: (i) systematically
characterize the structure, binding kinetics, and signaling potency of Notch receptor-ligand complexes; (ii) use
electron microscopy to visualize the ultrastructural architecture of full-length Notch1-DLL4 and Notch1-Jag1
complexes; (iii) determine the biophysical basis by which Notch glycosylation regulates DLL and Jag activity,
and (iv) design tissue-specific Notch antagonists. The successful completion of this work will establish the
molecular mechanisms of Notch receptor-ligand selectivity and will guide the engineering of novel modulators of
Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.

## Key facts

- **NIH application ID:** 10189660
- **Project number:** 5R35GM133482-03
- **Recipient organization:** H. LEE MOFFITT CANCER CTR & RES INST
- **Principal Investigator:** Vincent Christopher Luca
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $430,000
- **Award type:** 5
- **Project period:** 2019-09-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10189660

## Citation

> US National Institutes of Health, RePORTER application 10189660, Illuminating Notch receptor-ligand selectivity through structure-guided protein engineering (5R35GM133482-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10189660. Licensed CC0.

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