A Closed Form Model for Molecular Ratchet-Type Chemically Induced Dimerization Modules

Abstract

Chemical-induced dimerization (CID) modules enable users to implement ligand-controlled cellular and biochemical functions for a number of problems in basic and applied biology. A special class of CID modules occur naturally in plants and involve a hormone receptor that binds a hormone, triggering a conformational change in the receptor that enables recognition by a second binding protein. Two recent reports show that such hormone receptors can be engineered to sense dozens of structurally diverse compounds. As a closed form model for molecular ratchets would be of immense utility in forward engineering of biological systems, here we have developed a closed form model for these distinct CID modules. These modules, which we call molecular ratchets, are distinct from more common CID modules called molecular glues in that they engage in saturable binding kinetics and are characterized well by a Hill equation. A defining characteristic of molecular ratchets is that the sensitivity of the response can be tuned by increasing the molar ratio of the hormone receptor to the binding protein. Thus, the same molecular ratchet can have a pico-or micromolar EC 50 depending on the concentration of the different receptor and binding proteins. Closed form models are derived for a base elementary reaction rate model, for ligand-independent complexation of the receptor and binding protein, and for homodimerization of the hormone receptor. Useful governing equations for a variety of in vitro and in vivo applications are derived, including enzyme-linked immunosorbent assay-like microplate assays, transcriptional activation in prokaryotes and eukaryotes, and ligand-induced split protein complementation.

Rationalizing Diverse Binding Mechanisms to the Same Protein Fold: Insights for Ligand Recognition and Biosensor Design

“…The START domain superfamily member PYR1 has recently been engineered to recognize dozens of natural and synthetic cannabinoids, organophosphates, and fungicides with micromolar to picomolar EC50s. ,, PYR1, along with its binding partner HAB1, is part of a natural chemically induced dimerization (CID) system utilized by the plant hormone abscisic acid. , Engineered PYR1 binds its cognate ligand independently of HAB1, which then enables HAB1 recognition to form a ternary complex (Figure A) . This “molecular ratchet” architecture is particularly well-suited for biosensors because the same molecular recognition component can be coupled to many different output signals …”

“…Population measurements from library titrations were also further validated by a phosphatase inhibition assay with purified protein in vitro (Figure S2). While in vitro EC50 values and yeast measurements of effective dissociation constants cannot be quantitatively compared, 34 the relative order of variants is the same. The heatmaps in Figures 3 and 4 include a subset of the data encompassing the 23 ligand-facing positions and display the average of two replicates unless a value could be calculated from only one replicate.…”

“…32,33 Engineered PYR1 binds its cognate ligand independently of HAB1, which then enables HAB1 recognition to form a ternary complex (Figure 1A). 34 This "molecular ratchet" architecture is particularly well-suited for biosensors because the same molecular recognition component can be coupled to many different output signals. 1 Crystal structures of both the wild-type and several engineered sensors all show a similar gate-latch-lock mechanism, 13,35 in which a hydrogen bond acceptor on the bound ligand coordinates a key water molecule at the top of the binding pocket (Figure 1B).…”

Rationalizing diverse binding mechanisms to the same protein fold: insights for ligand recognition and biosensor design

“…PYR1, along with its binding partner HAB1, is part of a natural chemically induced dimerization (CID) system utilized by the plant hormone abscisic acid 32,33 . Engineered PYR1 binds its cognate ligand independently from HAB1, which then enables HAB1 recognition to form a ternary complex ( Figure 1A ) 34 . This ‘molecular ratchet’ architecture is particularly well-suited for biosensors because the same molecular recognition component can be coupled to many different output signals 1 .…”

A simeprevir-inducible molecular switch for the control of cell and gene therapies

“…An alternative approach is to start from a known protein:small molecule complex and identify specific protein binding partners by selection from phage display libraries of antibody (or antibody-like scaffold) proteins 9 – 11 or computational design 12 . Protein engineering and directed evolution have also been used to alter the ligand-binding specificity of the natural ABA-based CID, thus creating a variety of CID modules 13 – 15 . These systems have been used primarily in vitro, as biosensors, or to regulate protein proximity-dependent cellular processes such as cellular co-localization, chimeric antigen receptor-T cell (CAR T) activation, and expression of endogenous and exogenous genes.…”