Engineered Receptor Construct for Ligand Detection
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Solution Overview
Problem
Current engineered receptor polypeptide constructs lack modularity and customization for specific ligand detection and response, and are sensitive to force or stretch of the cell membrane, limiting their effectiveness in biological applications.
Innovation Solution
Development of a modular engineered receptor polypeptide construct with an extracellular ligand binding domain lacking a Notch regulatory region, featuring a flexible linker, an intramolecular peptide, a transmembrane domain with a γ-secretase cleavage site, and an intracellular effector domain, which allows specific ligand binding to trigger γ-secretase cleavage and release of the effector domain, enabling customizable cellular responses.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a Notch regulatory region (NRR) is included in the extracellular ligand binding domain, then the sensor can detect ligands, but the sensor becomes sensitive to force or stretch of the cell membrane, reducing measurement precision
Solution Approach 1:
The invention removes the Notch regulatory region (NRR) from the extracellular ligand binding domain. This extraction eliminates the problematic NRR that causes sensitivity to membrane force and stretch, while preserving the ligand binding capability through the engineered intramolecular peptide system.
2Adaptability or versatility
If the receptor construct is designed to be sensitive to membrane force or stretch, then it can respond to mechanical stimuli, but it loses specificity for ligand detection, reducing measurement precision
Solution Approach 1:
The invention introduces an intramolecular peptide as an intermediary element that binds to the ligand binding site and sterically blocks γ-secretase access. This peptide acts as a mediator that translates specific ligand binding into controlled effector domain release, providing ligand-specific responses independent of mechanical stimuli.
3Reliability
If the intramolecular peptide has high affinity binding to the ligand binding site, then the construct maintains a closed conformation that inhibits γ-secretase cleavage, but the cognate ligand must have even higher affinity to displace it, reducing productivity
Solution Approach 1:
The invention optimizes the binding affinity parameters of the intramolecular peptide to achieve balanced performance. The peptide is engineered with specific affinity characteristics that ensure stable closed conformation at baseline while allowing efficient displacement by cognate ligand, achieving both reliability and productivity.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The construct enables precise detection of small molecules, peptides, or proteins, generating specific genetic or protein output responses, such as gene activation or cleavage, while being insensitive to membrane force or stretch, enhancing the modularity and effectiveness of cellular biosensors.
Implementation Method 1
an intramolecular peptide that binds to the at least one ligand binding site in the extracellular ligand binding domain
Implementation Method 2
a transmembrane domain comprising at least one γ-secretase cleavage site
Data Source
AI summary
Described herein are methods and compositions related to a modular engineered receptor polypeptide construct and their use in methods to modulate the activity of a cell. In particular, the disclosure relates to an engineered receptor polypeptide comprising, in brief, (i) an extracellular ligand binding domain having at least one ligand binding site, (ii) an optional flexible polypeptide linker, (iii) an intramolecular peptide that binds to the at least one ligand binding site in the extracellular ligand binding domain, (iv) a transmembrane domain comprising at least one γ-secretase cleavage site, and (v) an intracellular effector domain, where the intramolecular peptide that serves to regulate the activity of the engineered receptor polypeptide. Other aspects relate to cells comprising the engineered receptor polypeptide, and nucleic acid sequence encoding the engineered receptor polypeptide.