Highly Specific DNA Recognition by a Designed Miniature Protein

Many DNA-binding proteins employ a short R-helix as their primary DNA recognition element.1-5 When removed from their natural contexts, however, most R-helices neither fold nor bind DNA with high affinity.6,7 Here we describe a strategy for the design of miniature proteins that present a solvent-exposed R-helix able to recognize DNA with high affinity and specificity. This strategy involves dissecting those R-helical residues required for DNA recognition from their native protein context and grafting them on a stable, miniature protein core (Figure 1). This strategy was used to design a 42-amino acid protein displaying the DNAcontact residues of a single basic region-leucine zipper (bZIP) protein. This miniature protein bound DNA in the low nanomolar concentration range under physiological solution conditions and exhibited greater sequence specificity than a natural bZIP protein. This strategy may represent a general approach to the design of small, folded proteins that recognize nucleic acid and protein targets with high affinity and specificity. Our design process began with avian pancreatic polypeptide (aPP), a small, well-folded protein of known structure consisting of a single R-helix stabilized by hydrophobic interaction with a type II polyproline helix (Figure 1).8-10 Since formation of the aPP core requires residues on only one face of the aPP R-helix (shown in blue), the opposite, solvent-exposed face of the R-helix is available for recognition of other macromolecular targets. By grafting various combinations of those thirteen residues (shown in pink) used by GCN4 to recognize the CRE half site (ATGAC, hsCRE)1,2 on the solvent-exposed R-helical face of aPP, we generated a series of polyproline helix-basic region (PPBRSR) molecules containing most or all of the DNA-contact residues of GCN4 and most or all of the folding residues of aPP (Figure 2). This procedure generated three positions of conflict, where essential DNA-contact and aPP-folding residues occupied a single position on the helix (Figure 2). No significant DNA binding was detected with peptides PPBR0SR, PPBR10SR, and PPBR11SR, which lacked one or more of these DNA-contact residues. Highaffinity DNA binding was observed with a peptide that contained these three residues: The equilibrium dissociation constant (Kd) of the PPBR2SR‚hsCRE complex was 5 nM under conditions of physiological ionic strength (Figure 3). DNA affinity was enhanced further by selective alanine substitutions that increased the overall R-helical propensity of the peptide, producing the PPBR4‚hsCRE24 complex whose Kd was 1.5 nM under identical conditions. Formation of the PPBR4‚hsCRE24 complex was unaffected by high concentrations of poly (dIdC)‚(dIdC) or a scrambled CRE site (NON), indicating that the high stability of PPBR4‚hsCRE24 was not due primarily to nonspecific ionic interactions. Circular dichroism experiments indicated that like bZIP peptides,6,11 PPBR4SR attained a fully R-helical conformation only in the presence of specific DNA.12 Although others have described monopartite DNA recognition by basic segment peptides, the affinities reported have been only moderate (60 nM-3 μM), and the complexes are stable only in very low ionic strength buffers.13,14 PPBR4SR represents the first example of high affinity, monopartite, major groove recognition at physiological ionic strength. To examine the contribution of hydrophobic core formation to PPBR4‚hsCRE24 complex stability, we studied peptides G27 * To whom correspondence should be addressed. (1) Ellenberger, T. E.; Brandl, C. J.; Struhl, K.; Harrison, S. C. Cell 1992, 71, 1223. (2) Konig, P.; Richmond, T. J. Mol. Biol. 1993, 233, 139. (3) Aggarwal, A. K.; Rodgers, D. W.; Drottar, M.; Ptashne, M.; Harrison, S. C. Science 1988, 242. (4) Ferre-D’Amare, A. R.; Prendergast, G. C.; Ziff, E. B.; Burley, S. K. Nature (London) 1993, 363, 38. (5) Kissinger, C. R.; Liu, B.; Martin-Blanco, E.; Kornberg, T. B.; Pabo, C. O. Cell 1990, 63, 579. (6) Weiss, M. A.; Ellenberger, T.; Wobbe, C. R.; Lee, J. P.; Harrison, S. C.; Struhl, K. Nature (London) 1990, 347, 575. (7) O’Neil, K. T.; Shuman, J. D.; Ampe, C.; DeGrado, W. F. Biochemistry 1991, 30, 9030. (8) Blundell, T. L.; Pitts, J. E.; Tickle, I. C.; Wood, S. P.; Wu, C.-W. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 4175. (9) Tonan, K.; Kawata, Y.; Hamaguchi, K. Biochemistry 1990, 29, 4424. (10) Hermans, J., Jr.; Scheraga, H. A. J. Am. Chem. Soc. 1961, 83, 3283. (11) O’Neil, K. T.; Hoess, R. H.; DeGrado, W. F. Science 1990, 249, 774. (12) The CD spectrum of PPBR4SR was unchanged between 1 and 20 μM, indicating that no detectable changes in secondary structure occurred in this range. Addition of hsCRE DNA significantly increased the R-helix content of PPBR4SR; smaller changes were observed upon addition of hsCEBP DNA (see Supporting Information). (13) Park, C.; Campbell, J. L.; Goddard, W. A., III J. Am. Chem. Soc. 1996, 118, 4235. (14) Morii, T.; Yamane, J.; Aizawa, Y.; Makino, K.; Sugiura, Y. J. Am. Chem. Soc. 1996, 118, 10011. Figure 1. Protein grafting strategy for the design of miniature DNAbinding proteins.