Orthogonal protein heterodimers

Abstract

Disclosed herein are designed heterodimer proteins, monomeric polypeptides capable of forming heterodimer proteins, protein scaffolds including such polypeptides, and methods for using the heterodimer proteins and subunit polypeptides for designing logic gates.

1 - 116 . (canceled) 
     
     
         117 . A cell comprising a designed protein logic gate, wherein:
 (a) the designed protein logic gate comprises:
 (i) one or more designed dimerizers, wherein:
 (A) each of the dimerizers comprises a fusion of two monomer subunits from different designed heterodimers connected by a polypeptide linker, wherein each of the monomer subunits comprises a binding interface; and 
 (B) each of the dimerizers comprises a plurality of hydrophobic residues forming substantially hydrophobic interaction surfaces that regulate the association of the binding interfaces with each other; 
 
 (ii) a first designed target monomer comprising:
 (A) a first effector polypeptide domain; and 
 (B) one or more subunits, each subunit comprising a binding interface; and 
 
 (iii) a second designed target monomer comprising:
 (A) a second effector polypeptide domain, and 
 (B) one or more subunits, each subunit comprising a binding interface; 
 
   (b) each binding interface of the one or more dimerizers is capable of a binding interaction via its binding interface to the binding interface of (i) another dimerizer, (ii) the first target monomer, or (iii) the second target monomer, wherein the interaction is by an asymmetric hydrogen bond network, to collectively and cooperatively effect gate activation and thereby effect control over a biological function of the cell.   
     
     
         118 . The cell of  claim 117  wherein the protein logic gate consists essentially of:
 (i) two dimerizers and the first and second designed target monomers; or 
 (iii) three dimerizers and the first and second designed target monomers. 
 
     
     
         119 . The cell of  claim 117  wherein in the absence of binding the first and/or second designed target monomers:
 (i) each of the one or more dimerizers is in a closed state wherein the hydrophobic interaction surfaces of such dimerizers are buried; and 
 (ii) the closed state is a folded state. 
 
     
     
         120 . The cell of  claim 119  wherein:
 (iii) the closed state establishes an energy barrier; and 
 (iv) energy provided by interactions of the one or more dimerizers with the first and second target monomers is sufficient to overcome the energy barrier to effect gate activation. 
 
     
     
         121 . The cell of  claim 117  wherein binding of the one or more dimerizers with the first or second designed target monomers occurs only in the presence of both the first and second designed target monomers. 
     
     
         122 . The cell of  claim 117  wherein:
 (i) the one or more dimerizers each comprises four alpha helices; and 
 (ii) in the absence of binding to the first and/or second target monomers, the binding interfaces of each of the one or more dimerizers are sequestered by hydrophobic interactions within the four helices. 
 
     
     
         123 . The cell of  claim 117  wherein the linker is a 6-residue linker or a 12-residue linker. 
     
     
         124 . The cell of  claim 117  wherein, the two monomer subunits from the different designed heterodimers comprise:
 (i) a first monomer subunit comprising a first alpha helix unit consisting essentially of one alpha helix; and 
 (ii) a second monomer subunit comprising a second alpha helix unit consisting essentially of three alpha helices. 
 
     
     
         125 . The cell of  claim 117  wherein, the two monomer subunits from the different designed heterodimers comprise:
 (i) a first monomer subunit comprising a first alpha helix unit consisting essentially of two alpha helices; and 
 (ii) a second monomer subunit comprising a second alpha helix unit consisting essentially of two alpha helices. 
 
     
     
         126 . The cell of  claim 117  wherein the first and/or second effector polypeptide domains are selected from the group consisting of nucleic acid binding proteins, transcription factors, receptor binding proteins, split enzymes, and effectors of membrane receptors. 
     
     
         127 . The cell of  claim 117  wherein the first and/or second effector polypeptide domains include an activation domain. 
     
     
         128 . The cell of  claim 117  wherein the first and/or second effector polypeptide domains include a DNA binding domain. 
     
     
         129 . The cell of  claim 117  wherein:
 (i) cooperative binding interactions involving all of the dimerizers and the first and second designed target monomers activate the gate to mediate the biological function of the cell; 
 or 
 (ii) cooperative binding interactions involving any one of the dimerizers with the first and second designed target monomers activates the gate to mediate the biological function of the cell. 
 
     
     
         130 . The cell of  claim 117  wherein:
 (i) the logic gate consists essentially of two of the dimerizers and the first and second designed target monomers; and 
 (ii) cooperative binding interactions involving both of the dimerizers and the first and second designed monomers activates the gate to mediate the biological function of the cell; 
 or 
 (iii) the logic gate consists essentially of three of the dimerizers and the first and second designed target monomers; and 
 (iv) cooperative binding interactions involving all three of the dimerizers and first and second designed monomers activates the gate to mediate the biological function of the cell.