Mutant g-protein coupled receptors and methods for selecting them

Abstract

The invention relates to mutant G-protein coupled receptors with increased conformational stability, and methods of use thereof. In some aspects, polynucleotides encoding the mutant G-protein coupled receptors are provided. In some aspects, host cells comprising the polynucleotides are provided. In some aspects, the invention relates to crystallized forms of the mutant G-protein coupled receptors, and methods of preparing the same.

1 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an agonist conformation compared to its parent GPCR in an agonist conformation,
 wherein the mutant GPCR has increased binding retention to an agonist ligand relative to its parent GPCR under denaturing conditions, and   wherein the mutant GPCR differs from its parent GPCR by one or more point mutations in a structural motif of the mutant GPCR selected from the group consisting of a helical interface, a helix kink, a helix opposite a helix kink, a helix surface pointing into the lipid bilayer, a helix surface pointing into the lipid bilayer at the hydrophobic-hydrophilic boundary layer, a loop region and a protein binding pocket.   
     
     
         2 . A mutant GPCR according to  claim 1 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         3 . A mutant GPCR according to  claim 2 , wherein the mutant GPCR has increased conformational thermostability, which is at least 1° C. more stable than its parent. 
     
     
         4 . (canceled) 
     
     
         5 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is a Class 1 GPCR. 
     
     
         6 .- 20 . (canceled) 
     
     
         21 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an antagonist conformation compared to its parent GPCR in an antagonist conformation,
 wherein the mutant GPCR has increased binding retention to an antagonist ligand relative to its parent GPCR under denaturing conditions, and   wherein the mutant GPCR differs from its parent GPCR by one or more point mutations in a structural motif of the mutant GPCR selected from the group consisting of a helical interface, a helix kink, a helix opposite a helix kink, a helix surface pointing into the lipid bilayer, a helix surface pointing into the lipid bilayer at the hydrophobic-hydrophilic boundary layer, a loop region and a protein binding pocket.   
     
     
         22 . A mutant GPCR according to  claim 21 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         23 . A mutant GPCR according to  claim 22 , wherein the mutant GPCR has increased conformational thermostability, which is at least 1° C. more stable than its parent. 
     
     
         24 . A mutant GPCR according to  claim 1 , wherein the structural motif is a helical interface or a helix surface pointing into the lipid bilayer. 
     
     
         25 . A mutant GPCR according to  claim 21 , wherein the structural motif is a helical interface or a helix surface pointing into the lipid bilayer. 
     
     
         26 . A mutant GPCR according to  claim 1 , which contains, compared to its parent GPCR, from 1 to 10 replaced amino acids. 
     
     
         27 . A mutant GPCR according to  claim 21 , which contains, compared to its parent GPCR, from 1 to 10 replaced amino acids. 
     
     
         28 . A mutant GPCR according to  claim 21 , wherein the mutant GPCR is a Class 1 GPCR. 
     
     
         29 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR is selected from the group consisting of: a mutant adenosine receptor, a mutant β-adrenergic receptor, a mutant neurotensin receptor, a mutant muscarinic acid receptor, a mutant 5-hydroxytryptamine receptor, a mutant adrenoceptor, a mutant anaphylatoxin receptor, a mutant angiotensin receptor, a mutant apelin receptor, a mutant bombesin receptor, a mutant bradykinin receptor, a mutant cannabinoid receptor, a mutant chemokine receptor, a mutant cholecystokinin receptor, a mutant dopamine receptor, a mutant endothelin receptor, a mutant free fatty acid receptor, a mutant bile acid receptor, a mutant galanin receptor, a mutant motilin receptor, a mutant ghrelin receptor, a mutant glycoprotein hormone receptor, a mutant GnRH receptor, a mutant histamine receptor, a mutant KiSS1-derived peptide receptor, a mutant leukotriene and lipoxin receptor, a mutant lysophospholipid receptor, a mutant melanin-concentrating hormone receptor, a mutant melanocortin receptor, a mutant melatonin receptor, a mutant neuromedin U receptor, a mutant neuropeptide receptor, a mutant N-formylpeptide family receptor, a mutant nicotinic acid receptor, a mutant opiod receptor, a mutant op sin-like receptor, a mutant orexin receptor, a mutant P2Y receptor, a mutant peptide P518 receptor, a mutant platelet-activating factor receptor, a mutant prokineticin receptor, a mutant prolactin-releasing peptide receptor, a mutant prostanoid receptor, a mutant protease-activated receptor, a mutant relaxin receptor, a mutant somatostatin receptor, a mutant SPC/LPC receptor, a mutant tachykinin receptor, a mutant trace amino receptor, a mutant thryotropin-releasing hormone receptor, a mutant urotensin receptor, a mutant vasopressin/oxytocin receptor, a mutant orphan GPCR, a mutant calcitonin receptor, a mutant corticotropin releasing factor receptor, a mutant glucagon receptor, a mutant parathyroid receptor, a mutant VIP/PACAP receptor, a mutant LNB7TM receptor, a mutant GABA receptor, a mutant metabotropic glutamate receptor, and a mutant calcium sensor receptor. 
     
     
         30 . A mutant GPCR according to  claim 1 , wherein the mutant GPCR has, compared to its parent GPCR, at least one different amino acid at a position which corresponds to any one or more of the following positions: (i) according to the numbering of the turkey β-adrenergic receptor as set out in  FIG. 9 : Ile 55, Gly 67, Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly 197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala 334, Phe 338, (ii) according to the numbering of the human adenosine A2a receptor as set out in  FIG. 10 : Gly 114, Gly 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg 220, Ser 223, Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315, (iii) according to the numbering of the rat neurotensin receptor as set out in  FIG. 11 : Ala 69, Leu 72, Ala 73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile 116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys 176, Ala 177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, Gly 209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn 262, Val 268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val 309, Leu 310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Val 360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro 389, Gly 390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399, and (iv) according to the numbering of the muscarinic receptor as set out in  FIG. 17 : Leu 65, Met 145, Leu 399, Ile 383 and Met 384. 
     
     
         31 . A mutant GPCR according to  claim 1 , wherein (i) the receptor is a mutant β-adrenergic receptor which, when compared to the corresponding wild-type adrenergic receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the turkey β-adrenergic receptor as set out in  FIG. 9 : Ile 55, Gly 67, Arg 68, Val 89, Met 90, Gly 98, Ile 129, Ser 151, Val 160, Gln 194, Gly 197, Leu 221, Tyr 227, Arg 229, Val 230, Ala 234, Ala 282, Asp 322, Phe 327, Ala 334, Phe 338; or wherein (ii) the receptor is a mutant adenosine receptor which, when compared to the corresponding wild-type adenosine receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the human adenosine A2a receptor as set out in  FIG. 10 : Gly 114, Gly 118, Leu 167, Ala 184, Arg 199, Ala 203, Leu 208, Gln 210, Ser 213, Glu 219, Arg 220, Ser 223, Thr 224, Gln 226, Lys 227, His 230, Leu 241, Pro 260, Ser 263, Leu 267, Leu 272, Thr 279, Asn 284, Gln 311, Pro 313, Lys 315; or wherein (iii) the receptor is a mutant neurotensin receptor which, when compared to the corresponding wild-type neurotensin receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the rat neurotensin receptor as set out in  FIG. 11 : Ala 69, Leu 72, Ala 73, Ala 86, Ala 90, Ser 100, His 103, Ser 108, Leu 109, Leu 111, Asp 113, Ile 116, Ala 120, Asp 139, Phe 147, Ala 155, Val 165, Glu 166, Lys 176, Ala 177, Thr 179, Met 181, Ser 182, Arg 183, Phe 189, Leu 205, Thr 207, Gly 209, Gly 215, Val 229, Met 250, Ile 253, Leu 256, Ile 260, Asn 262, Val 268, Asn 270, Thr 279, Met 293, Thr 294, Gly 306, Leu 308, Val 309, Leu 310, Val 313, Phe 342, Asp 345, Tyr 349, Tyr 351, Ala 356, Phe 358, Val 360, Ser 362, Asn 370, Ser 373, Phe 380, Ala 385, Cys 386, Pro 389, Gly 390, Trp 391, Arg 392, His 393, Arg 395, Lys 397, Pro 399; or wherein (iv) the receptor is a mutant muscarinic receptor which, when compared to the corresponding wild-type muscarinic receptor, has a different amino acid at a position which corresponds to any one or more of the following positions according to the numbering of the human muscarinic receptor as set out in  FIG. 17 : Leu 65, Met 145, Leu 399, Ile 383 and Met 384. 
     
     
         32 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an agonist conformation compared to its parent GPCR in an agonist conformation, produced by a method comprising:
 (a) preparing one or more mutants of a parent GPCR, wherein the one or more mutants each differs from its parent GPCR by one or more point mutations and wherein the one or more mutants and the parent GPCR are provided in a solubilised form obtained by solubilising the one or more mutants and the parent GPCR from a membrane using a detergent;   (b) contacting the one or more mutants and the parent GPCR with an agonist ligand to select for one or more mutants with increased conformational stability in the agonist conformation;   (c) exposing to denaturing conditions the one or more mutants and the parent GPCR, wherein the one or more mutants and the parent GPCR are bound to the ligand;   (d) measuring the stability of binding of the one or more mutants and the parent GPCR to the ligand by measuring the ability of the one or more mutants and the parent GPCR to retain ligand binding capacity following exposure to denaturing conditions; and   (e) selecting a mutant GPCR that exhibits increased stability of binding to the ligand following exposure to denaturing conditions.   
     
     
         33 . A mutant G-protein coupled receptor (GPCR) with increased conformational stability in an antagonist conformation compared to its parent GPCR in an antagonist conformation, produced by a method comprising:
 (a) preparing one or more mutants of a parent GPCR, wherein the one or more mutants each differs from its parent GPCR by one or more point mutations and wherein the one or more mutants and the parent GPCR are provided in a solubilised form obtained by solubilising the one or more mutants and the parent GPCR from a membrane using a detergent;   (b) contacting the one or more mutants and the parent GPCR with an antagonist ligand to select for one or more mutants with increased conformational stability in the antagonist conformation;   (c) exposing to denaturing conditions the one or more mutants and the parent GPCR, wherein the one or more mutants and the parent GPCR are bound to the ligand;   (d) measuring the stability of binding of the one or more mutants and the parent GPCR to the ligand by measuring the ability of the one or more mutants and the parent GPCR to retain ligand binding capacity following exposure to denaturing conditions; and   (e) selecting a mutant GPCR that exhibits increased stability of binding to the ligand following exposure to denaturing conditions.   
     
     
         34 . A mutant GPCR according to  claim 32 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         35 . A mutant GPCR according to  claim 33 , wherein the denaturing conditions are selected from the group consisting of heat, a detergent, a chaotropic agent and an extreme of pH. 
     
     
         36 . A mutant GPCR according to  claim 32 , wherein the mutant GPCR is a Class 1 GPCR. 
     
     
         37 . A mutant GPCR according to  claim 33 , wherein the mutant GPCR is a Class 1 GPCR.