title: Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.
abstract: The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors.forename: Jenifer B
authors.initials: JB
authors.lastname: Kaplan
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Reinke
authors.forename: Amy E
authors.initials: AE
authors.lastname: Keating
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D015195:Drug Design; D006801:Humans; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D055672:Static Electricity
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D010455:Peptides
keywords: bZIP transcription factors; coiled coil; helix propensity; protein design; protein-protein interactions
references.citation: Annu Rev Cell Dev Biol. 2012;28:251-77
references.pmid: 23057742
references.citation: Cell. 1992 Feb 21;68(4):699-708
references.pmid: 1739975
references.citation: Genes Dev. 1993 Jun;7(6):1047-58
references.pmid: 8504929
references.citation: Neuron. 1995 May;14(5):927-39
references.pmid: 7748560
references.citation: Biophys J. 1997 Jan;72(1):65-76
references.pmid: 8994593
references.citation: Protein Sci. 2012 Sep;21(9):1241-52
references.pmid: 22811394
references.citation: Biochemistry. 2009 Nov 3;48(43):10380-8
references.pmid: 19743874
references.citation: Structure. 2002 Sep;10(9):1235-48
references.pmid: 12220495
references.citation: Science. 2003 Jun 27;300(5628):2097-101
references.pmid: 12805554
references.citation: J Mol Biol. 2000 Jul 28;300(5):1377-87
references.pmid: 10903875
references.citation: Biochemistry. 2006 Sep 26;45(38):11324-32
references.pmid: 16981692
references.citation: EMBO J. 1995 Nov 1;14(21):5329-37
references.pmid: 7489722
references.citation: Biochemistry. 1993 Jun 1;32(21):5491-6
references.pmid: 8504069
references.citation: Int J Biochem Cell Biol. 2008;40(1):14-21
references.pmid: 17466566
references.citation: J Mol Biol. 1994 Apr 8;237(4):500-12
references.pmid: 8151708
references.citation: J Mol Biol. 2006 Feb 3;355(5):1125-42
references.pmid: 16359704
references.citation: Biochemistry. 2006 Apr 11;45(14):4463-73
references.pmid: 16584182
references.citation: BMC Evol Biol. 2005 Nov 16;5:66
references.pmid: 16288662
references.citation: J Am Chem Soc. 2007 Mar 7;129(9):2456-7
references.pmid: 17284038
references.citation: Structure. 2005 Feb;13(2):225-34
references.pmid: 15698566
references.citation: Nature. 1986 Jan 16-22;319(6050):199-203
references.pmid: 3945310
references.citation: Biophys J. 2002 Feb;82(2):1096-111
references.pmid: 11806949
references.citation: Biochemistry. 2007 Apr 24;46(16):4804-14
references.pmid: 17402748
references.citation: Mol Cell. 2011 Jul 8;43(1):145-55
references.pmid: 21726817
references.citation: J Mol Biol. 2011 Oct 14;413(1):222-35
references.pmid: 21854787
references.citation: J Mol Biol. 1998 Nov 20;284(1):173-91
references.pmid: 9811549
references.citation: Nat Struct Biol. 2003 Jan;10(1):45-52
references.pmid: 12459719
references.citation: J Mol Biol. 1998 Jun 19;279(4):959-72
references.pmid: 9642074
references.citation: PLoS Comput Biol. 2009 Dec;5(12):e1000627
references.pmid: 20041208
references.citation: Nucleic Acids Res. 2002 May 15;30(10):e43
references.pmid: 12000848
references.citation: Mol Cell Biol. 2002 Sep;22(18):6321-35
references.pmid: 12192032
references.citation: Biochemistry. 2002 Dec 3;41(48):14122-31
references.pmid: 12450375
references.citation: Protein Sci. 2012 Jul;21(7):949-63
references.pmid: 22593041
references.citation: EMBO J. 1994 Jun 15;13(12):2849-61
references.pmid: 8026470
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):8989-94
references.pmid: 16754880
references.citation: Science. 2013 May 10;340(6133):730-4
references.pmid: 23661758
references.citation: Biochemistry. 2010 Mar 9;49(9):1985-97
references.pmid: 20102225
references.citation: Genome Biol. 2004;5(2):R11
references.pmid: 14759261
references.citation: ACS Synth Biol. 2012 Apr 20;1(4):118-29
references.pmid: 22558529
references.citation: J Mol Biol. 2011 Apr 29;408(2):304-20
references.pmid: 21354428
references.citation: Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129-57
references.pmid: 1751545
references.citation: Cell. 2010 Mar 5;140(5):744-52
references.pmid: 20211142
references.citation: Dev Dyn. 2004 Mar;229(3):440-8
references.pmid: 14991699
references.citation: Adv Protein Chem. 2003;66:27-85
references.pmid: 14631816
references.citation: Protein Sci. 2002 Nov;11(11):2714-26
references.pmid: 12381853
references.citation: J Am Chem Soc. 2008 Sep 10;130(36):12038-44
references.pmid: 18698842
references.citation: Genes Dev. 1998 Oct 15;12(20):3168-81
references.pmid: 9784492
references.citation: Nature. 2009 Apr 16;458(7240):859-64
references.pmid: 19370028
references.citation: Mol Cell Biol. 1998 Feb;18(2):967-77
references.pmid: 9447994
references.citation: Genes Dev. 1988 Dec;2(12B):1687-99
references.pmid: 2467839
references.citation: J Biol Chem. 1997 Jul 25;272(30):18586-94
references.pmid: 9228025
references.citation: Curr Biol. 1993 Oct 1;3(10):658-67
references.pmid: 15335856
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 6;102(36):12724-9
references.pmid: 16129838
references.citation: Protein Sci. 1993 Jul;2(7):1072-84
references.pmid: 8102921
references.citation: Biopolymers. 1991 Nov;31(13):1463-70
references.pmid: 1814498
references.citation: J Mol Biol. 2008 Feb 8;376(1):288-301
references.pmid: 18177896
references.citation: Science. 1990 Nov 2;250(4981):646-51
references.pmid: 2237415
references.citation: Biochemistry. 1992 Jun 30;31(25):5739-46
references.pmid: 1610823
references.citation: Nat Struct Biol. 1994 Jun;1(6):399-409
references.pmid: 7664054
references.citation: Protein Sci. 1997 Jun;6(6):1333-7
references.pmid: 9194194
references.citation: J Mol Biol. 2000 Mar 3;296(4):1105-16
references.pmid: 10686107