Chimeric ABC Transporter Screening for Outward-Facing Inhibitors
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Solution Overview
Problem
Existing high-throughput screening methods for identifying molecules that inhibit ABC transporters, such as MsbA in Gram-negative bacteria, are biased towards inward-facing conformations, making it difficult to find molecules that bind to the outward-facing periplasmic, extracellular, and/or luminal face, which are solvent-accessible and can act as effective inhibitors without crossing multiple membranes.
Innovation Solution
Development of chimeric ABC transporter proteins where regions of the periplasmic, extracellular, and/or luminal face are substituted with equivalent regions from different ABC transporters, allowing for the identification of molecules that bind to the outward-facing conformation by using a counter-selection screen where molecules that bind to the parental but not the chimeric transporter are identified.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput screening methods use mutant cell types with more permeable outer membranes to identify molecules binding to inward-facing conformation, then screening can be performed, but the identified molecules are biased toward inward-facing conformation and most cannot cross membranes to be effective inhibitors in wild-type cells
Solution Approach 1:
The patent inverts the conventional screening approach by using chimeric ABC transporters with swapped periplasmic loop regions. This allows the screening to selectively identify molecules that bind to outward-facing conformation at the periplasmic face, rather than relying on permeable membranes to access inward-facing conformations. The chimera design creates a selective pressure that favors molecules binding to the outward-facing state, reversing the traditional bias toward inward-facing binders.
Solution Approach 2:
The chimeric ABC transporter acts as an intermediary tool that mediates between the screening process and the target protein. By incorporating specific periplasmic loop regions from different ABC transporters, the chimera creates a controlled interface that selectively permits binding of outward-facing molecules while maintaining the structural framework needed for screening. This intermediary design filters out non-specific binders and focuses identification on molecules with specific outward-facing binding modes.
2Reliability
If molecules bind to the inward-facing conformation in Gram-negative cells, then binding can be detected, but the molecules must cross both outer membrane and inner membrane which most identified molecules cannot do
Solution Approach 1:
The patent reverses the conventional approach by targeting outward-facing conformations at the periplasmic face rather than inward-facing conformations requiring membrane penetration. The chimeric transporter design with swapped periplasmic loops creates a binding interface accessible from the periplasm, eliminating the need for molecules to cross multiple membranes while maintaining reliable binding detection. This inversion of the binding face fundamentally resolves the permeability barrier.
3Measurement precision
If chimeric ABC transporters are created with substituted periplasmic loop regions, then selective binding to outward-facing conformation can be achieved, but the device complexity increases
Solution Approach 1:
The patent applies segmentation by focusing the chimera construction on specific periplasmic loop regions rather than redesigning the entire ABC transporter. By swapping only the periplasmic loop segments (regions connecting transmembrane helices) while maintaining the core transmembrane structure and nucleotide-binding domains, the complexity is localized to specific functional segments. This modular approach achieves conformation-specific binding without requiring complete protein redesign.
Solution Approach 2:
The patent implements local quality by making specific changes only to the periplasmic loop regions of the ABC transporter while leaving the transmembrane domains and nucleotide-binding sites intact. The periplasmic loops are the only regions with altered sequence and structure in the chimera, creating localized functional differences that confer outward-facing binding specificity. This localized modification strategy achieves precise binding control with minimal overall structural disruption.
Data Source
AI summary
The present disclosure relates to chimeric ABC transporter proteins and methods of screening for molecules that bind to the periplasmic, extracellular, and/or luminal face of an ABC transporter protein using the chimeric ABC transporters. For example, in some embodiments, screening methods involve providing a chimeric ABC transporter in which one or more regions of the periplasmic, extracellular, and/or luminal face of the ABC transporter are substituted with one or more equivalent regions of the periplasmic, extracellular, and/or luminal face of a different ABC transporter and selecting for molecules that bind to the ABC transporter but do not bind to the chimeric ABC transporter. The disclosure also relates to molecules that bind to the periplasmic, extracellular, and/or luminal face of an ABC transporter protein, for example, identified in such screens.