Angewandte
Chemie
than 58 were accepted into the final ensemble. A total of 64 dihedral-
angle restraints were obtained from backbone chemical shifts by using
TALOS.[26] Furthermore, 14 hydrogen-bond restraints were identified
from the NOE pattern in the 3D 13C-edited NOESY spectrum.
15N-labeled AF6 PDZ at 50 mM concentration in a 20 mm Na
phosphate buffer solution containing NaCl (50 mm), and [D]6DMSO
(10% v/v) at pH 7.0 was used for screening experiments. Ligand
binding was detected at 300 K by acquiring 1H-15N HSQC spectra in
the presence and absence of compounds. Compounds were initially
tested at 400 mm each in mixtures of 16 compounds, with subsequent
deconvolution to mixtures of 4 compounds at 400 mm each and then to
individual compounds. Spectra were acquired with 16 scans and 128
points in the indirect dimension on a Bruker DRX600 spectrometer
equipped with a cryoprobe. Chemical-shift mapping of the binding
site was achieved by comparing the shifts of protein alone to those of
[15] G. Bruno, L. Constantino, C. Curinga, R. Maccari, F. Manforte,
F. Nicolꢂ, R. Ottanꢃ, M. G. Vigorita, Bioorg. Med. Chem. 2002,
10, 1077 – 1084.
[16] S. Kukolya, S. Draheim, B. Graves, D. Hunden, J. Pfeil, R.
Cooper, J. Ott, F. Counter, J. Med. Chem. 1985, 28, 1896 – 1903.
[17] N. L. Stricker, K. S. Christopherson, B. A. Yi, P. J. Schatz, R. W.
Raab, G. Dawes, D. E. Bassett Jr, D. S. Bredt, M. Li, Nat.
Biotechnol. 1997, 15, 336 – 342.
[18] R. A. Laskowski, J. A. Rullmann, M. W. MacArthur, R. Kap-
tein, J. M. Thornton, J. Biomol. NMR 1996, 8, 477 – 486.
[19] R. Koradi, M. Billeter, K. Wꢄthrich, J. Mol. Graphics 1996, 14,
29 – 32.
[20] D. A. Case, T. A. Darden, T. E. Cheatham III, C. L. Simmerling,
J. Wang, R. E. Duke, R. Luo, K. M. Merz, B. Wang, D. A.
Pearlman, M. Crowley, S. Brozell, V. Tsui, H. Gohlke, J.
Mongan, V. Hornak, G. Cui, P. Beroza, C. Schafmeister, J. W.
Caldwell, W. S. Ross, P. A. Kollman, AMBER 8, University of
California, San Francisco, 2004.
the protein in presence of ligands. Chemical shifts were quantified by
1
using the formula Dd = [(DH)2 + (DN/5)2] = , in which Dd is the
2
weighted chemical-shift change, and DH and DN are the chemical
shift changes in the proton and the nitrogen dimensions, respectively.
Dissociation constants were obtained for selected compounds by
monitoring the chemical shift changes as a function of ligand
concentration. Data were fit by using a one-site binding model. A
nonlinear least-square optimization was performed by varying the
values of Kd and the chemical shift of the fully saturated protein.
[21] M. S. Wallace, R. A. Laskowski, J. M. Thornton, Protein Eng.
1995, 8, 127 – 134.
[22] G. Birrane, J. Chung, J. A. Ladias, J. Biol. Chem. 2003, 278,
1399 – 1402.
[23] V. Kanelis, J. D. Forman-Kay, L. E. Kay, IUBMB Life 2001, 52,
291 – 302.
[24] T. D. Goddard, D. G. Kneller, SPARKY 3, University of
California, San Francisco 2003.
[25] P. Guntert, C. Mumenthaler, K. Wꢄthrich, J. Mol. Biol. 1997,
273, 283 – 298.
Received: November 8, 2005
Revised: January 26, 2006
Published online: May 3, 2006
[26] G. Cornilescu, F. Delaglio, A. Bax, J. Biomol. NMR 1999, 13,
289 – 302.
Keywords: inhibitors · ligand effects · NMR spectroscopy ·
PDZ domains · protein structures
.
[1] T. Pawson, P. Nash, Science 2003, 300, 445 – 452.
[2] Z. Songyang, A. S. Fanning, C. Fu, J. Xu, S. M. Marfatia, A. H.
Chishti, A. Crompton, A. C. Chan, J. M. Anderson, L. C.
Cantley, Science 1997, 275, 73 – 77.
[3] B. Z. Harris, W. A. Lim, J. Cell Sci. 2001, 114, 3219 – 3231.
[4] U. Wiedemann, P. Boisguerin, R. Leben, D. Leitner, G. Krause,
K. Moelling, R. Volkmer-Engert, H. Oschkinat, J. Mol. Biol.
2004, 343, 703 – 718.
[5] B. J. Hillier, K. S. Christopherson, K. E. Prehoda, D. S. Bredt,
W. A. Lim, Science 1999, 284, 812 – 815.
[6] D. A. Doyle, A. Lee, J. Lewis, E. Kim, M. Sheng, R. MacKinnon,
Cell 1996, 85, 1067 – 1076.
[7] M. Cabral, J. H. Petosa, M. J. Sutcliffe, S. Raza, O. Byron, F. Poy,
S. M. Marfatia, A. H. Chishti, R. C. Liddington, Nature 1996,
382, 649 – 652.
[8] J. Schultz, U. Hoffmuller, G. Krause, J. Ashurst, M. J. Macias, P.
Schmieder, J. Schneider-Mergener, H. Oschkinat, Nat. Struct.
Biol. 1998, 5, 19 – 24.
[9] R. Prasad, Y. Gu, H. Alder, T. Nakamura, O. Canaani, H. Saito,
K. Huebner, R. P. Gale, P. C. Nowell, K. Kuriyama, Cancer Res.
1993, 53, 5624 – 5628.
[10] B. Hock, B. Bꢁhme, T. Karn, T. Yamamoto, K. Kaibuchi, U.
Holtrich, S. Holland, T. Pawson, H. Rubsamen-Waigmann, K.
Strebhardt, Proc. Natl. Acad. Sci. USA 1998, 95, 9779 – 9784.
[11] M. Buchert, S. Schneider, V. Meskenaite, M. T. Adams, E.
Canaani, T. Baechi, K. Moelling, C. M. Hovens, J. Cell Biol.
1999, 144, 361 – 371.
[12] K. Takahashi, H. Nakanishi, M. Miyahara, K. Mandai, K. Satoh,
A. Satoh, H. Nishioka, J. Aoki, A. Nomoto, A. Mizoguchi, Y.
Takai, J. Cell Biol. 1999, 145, 539 – 549.
[13] K. Ebnet, C. U. Schulz, M. K. Meyer Zu Brickwedde, G. G.
Pendl, D. Vestweber, J. Biol. Chem. 2000, 275, 27979 – 27988.
[14] G. Radziwill, R. A. Erdmann, U. Margelisch, K. Moelling, Mol.
Cell. Biol. 2003, 23, 4663 – 4672.
Angew. Chem. Int. Ed. 2006, 45, 3790 –3795ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3795