Designing Protein Dimerizers: The Importance of Ligand Conformational Equilibria Jonathan C. T. Carlson, ² Aaron Kanter, ² Guruvasuthevan R. Thuduppathy, ² Vivian Cody, § Pamela E. Pineda, ² R. Scott McIvor, ‡ and Carston R. Wagner* ,² Department of Medicinal Chemistry, College of Pharmacy, and Department of Cell Biology, Genetics and DeVelopment, UniVersity of Minnesota, Minneapolis, Minnesota 55455, and Hauptman-Woodward Medical Research Institute Inc., 73 High St., Buffalo, New York 14203 Received March 21, 2002 ; E-mail: wagne003@tc.umn.edu Abstract: In an effort to elucidate the role of ligand conformation in induced protein dimerization, we synthesized a flexible methotrexate (MTX) dimer, demonstrated its ability to selectively dimerize Escherichia coli dihydrofolate reductase (DHFR), and evaluated the factors regulating its ability to induce cooperative dimerization. Despite known entropic barriers, bis-MTX proved to possess substantial conformational stability in aqueous solution (-3.8 kcal/mol g ΔGfold g -4.9 kcal/mol), exerting a dominant influence on the thermodynamics of dimerization. To dimerize DHFR, bis-MTX must shift from a folded to an extended conformation. From this conclusion, the strength of favorable protein-protein interactions in bis-MTX-E. coli DHFR dimers (-3.1 kcal/mol g ΔGc g -4.2 kcal/mol), and the selectivity of dimerization for E. coli DHFR relative to mouse DHFR (>10 7 ) could be determined. The crystal structure of bis-MTX in complex with E. coli DHFR confirms the feasibility of a close-packed dimerization interface and suggests a possible solution conformation for the induced protein dimers. Consequently, the secondary structure of this minimal foldamer regulates its ability to dimerize dihydrofolate reductase in solution, providing insight into the complex energy landscape of induced dimerization. Introduction Biological inducers (or modulators) of protein dimerization, such as erythropoeitin (EPO) and human growth hormone, regulate signal transduction, transcription, and metabolic pro- cesses. 1 Mirroring these functions, chemical inducers of protein dimerization (CIDs) have been used to control gene expres- sion, 2,3 modulate cell membrane receptor signaling, 4 selectively antagonize cellular processes, 5-7 search for novel biocatalysts, 8,9 and even to target protein heterodimers with no established ligand. 10 Moreover, CID-based systems may be clinically valuable as a tool to selectively regulate gene expression in cell- based therapies. 11 Despite these diverse applications, regulatory mechanisms governing chemically induced dimerization have been incompletely investigated. Parallels have been drawn to the diverse forms of conformational control that are a hallmark of biological regulation, essential in macromolecular recognition and signal transduction. Receptor conformation, for instance, has recently proven to be crucial in regulating activation of the dimerized EPO receptor, 12,13 and the behavior of synthetic ligands within this model analyzed. 14 Although the role of receptor conformation in these processes is now appreciated, the potentially significant influence of ligand conformationsin both biological and chemical casesshas been largely neglected. Dimerization exhibits a sensitive interplay between confor- mational and binding energetics, as increasing ligand concentra- tions compete for binding to dimerized proteins and ultimately favor monomerization. 15,16 It can be shown, first, that the association constant for binding of ligand to monomeric receptor, K 1 , determines the ligand concentration (K d ) 1/K 1 ) at which peak dimerization is observed. Second, the association constant * Corresponding author address: Department of Medicinal Chemistry, University of Minnesota, 8-174 Weaver Densford Hall, 308 Harvard St. SE, Minneapolis, MN 55455. Phone: 612 625-2614. ² Department of Medicinal Chemistry, College of Pharmacy. ‡ Department of Cell Biology, Genetics and Development. § Hauptman-Woodward Medical Research Institute Inc. (1) Klemm, J. D.; Schreiber, S. L.; Crabtree, G. R. Annu. ReV. Immunol. 1998, 16, 569-592. (2) Diver, S. T.; Schreiber, S. L. J. Am. Chem. Soc. 1997, 119, 5106-5109. (3) Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.; Keenan, T.; Natesan, S.; Pollock, R.; Yang, W.; Courage, N. L.; Holt, D. A.; Gilman, M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10618-10623. (4) Spencer, D. M.; Wandless, T. J.; Schreiber, S. L.; Crabtree, G. R. Science 1993, 262, 1019-1024. (5) Briesewitz, R.; Ray, G. T.; Wandless, T. J.; Crabtree, G. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1953-1958. (6) Lin, Y.-M.; Braun, P. D.; Ray, G. T.; Wandless, T. J. In Abstracts of Papers, 221st National Meeting of the American Chemical Socety, San Diego, CA, 2001; American Chemical Society: Washington, DC, 2001. (7) Barglow, K. T.; Lin, Y.-M.; Braun, P. D.; Wandless, T. J. Abstracts of Papers, 223rd National Meeting of the American Chemical Socety, Orlando, FL, 2002; American Chemical Society: Washington, DC, 2002. (8) Lin, H.; Abida, W. M.; Sauer, R. T.; Cornish, V. W. J. Am. Chem. Soc. 2000, 122, 4247-4248. (9) Firestine, S. M.; Salinas, F.; Nixon, A. E.; Baker, S. J.; Benkovic, S. J. Nature Biotech. 2000, 18, 544-547. (10) Koide, K.; Finkelstein, J. M.; Ball, Z.; Verdine, G. L. J. Am. Chem. Soc. 2001, 123, 398-408. (11) Neff, T.; Blau, C. A. Blood 2001, 97, 2535-2540. (12) Livnah, O.; Stura, E. A.; Middleton, S. A.; Johnson, D. L.; Jolliffe, L. K.; Wilson, I. A. Science 1999, 283, 987-990. (13) Remy, I.; Wilson, I. A.; Michnick, S. W. Science 1999, 283, 990-993. (14) Boger, D. L.; Goldberg, J. Bioorg. Med. Chem. 2001, 9, 557-562. (15) Perelson, A. S.; DeLisi, C. Math. Biosci. 1980, 48, 71-110. (16) Whitty, A.; Borysenko, C. W. Chem. Biol. 1999, 6, R107-R118. Published on Web 01/17/2003 10.1021/ja026264y CCC: $25.00 © 2003 American Chemical Society J. AM. CHEM. SOC. 2003, 125, 1501-1507 9 1501