Enzyme–Inhibitor Complex DOI: 10.1002/ange.200705614 Dynamics in the p38a MAP Kinase–SB203580 Complex Observed by Liquid-State NMR Spectroscopy** ValerieS.Honndorf,NicolasCoudevylle,StefanLaufer,StefanBecker,andChristianGriesinger* Diaryl-heterocycle compounds were the first small-molecule inhibitors targeting mitogen-activated protein (MAP) kin- ases. [1] The diaryl heterocycle SB203580 binds in an adenosine triphosphate (ATP) competitive manner to inactive and active p38a MAP kinase with similar IC 50 values. [2] A dissociation constant (K D ) of 11.5 nm was reported for the inactive form. [3] Crystallographic studies showed that SB203580 binds in the ATP-binding site of p38a. Similarly to ATP, the pyridine nitrogen atom of SB203580 forms a hydrogen bond to the backbone amide of Met109 from the hinge region. [4] In contrast to the binding of ATP, the positively charged side chain NH 3 + group of Lys53 forms a hydrogen bond to the imidazole nitrogen atom of SB203580, the fluorophenyl group is situated in the hydrophobic pocket I, and the methylsulfinyl group contacts the phosphate binding region below the glycine-rich loop while the linked phenyl ring forms stacking interactions with Tyr35 in the glycine-rich loop (Figure 1). [4,5] In the crystal structure (PDB code: 1A9U), the interact- ing residues are fully occupied with rather low B factors. This clearly suggests the formation of a very rigid complex between the inhibitor and the protein. Similar observations are made for the crystal structures of several other small- molecule inhibitor complexes of p38a as well as of the free form of the protein. [5,6] The hinge region seems to be the most important anchor point for diaryl-heterocycle kinase inhib- itors and its sequence is very specific for each MAP kinase. [7] To obtain a deeper insight into the effect of the conserved pharmacophore of these inhibitors on the dynamics of their interaction with inactive p38a in solution, we performed NMR measurements on the prototypical SB203580/p38a MAP kinase complex. [8] To verify the assignment of the free form of p38a [9] and to assign the p38a/SB203580 complex, we recorded a TROSY- HNCA spectrum for triple-labeled samples. As reported before, [10] only 75% of the expected resonances were observable, probably because of intrinsic dynamic heteroge- neity. About 70% of the observable resonances were assigned for both forms. Comparison of the 1 H– 15 N TROSY spectra of p38a in its free and inhibitor-bound forms revealed chemical- shift perturbation for a limited set of residues, all located near the binding site of SB203580 in the crystal structure (Figure 2), thereby confirming previous results. [10] To detect potential conformational changes induced by SB203580 bound to p38a in solution, we measured one-bond 1 D1 H– 15 N residual dipolar couplings (RDCs) for p38a in the free and inhibitor-bound forms in a weakly oriented medium (with 20mgmL À1 bacteriophage Pf1). The large size of p38a means that the measurement of RDCs leads to large errors. Only RDC values with an error lower than 5 Hz [11] were taken into account; this resulted in an ensemble of 73 couplings for the free form and 43 couplings for the inhibitor-bound form, which exhibits more dynamic heterogeneity. Each set of couplings was used to determine the alignment tensor by using the singular-value decomposition approach with the Figure 1. Structure of p38a in a complex with SB203580 (Protein Data Bank (PDB) access code: 1A9U). SB203580, as well as residues involved in the interaction, are represented as sticks; hydrogen bonds are represented as blue dashed lines. [*] Dipl.-Chem. V.S. Honndorf, Dr. N. Coudevylle, Dr. S. Becker, Prof.Dr. C. Griesinger Department of NMR-based Structural Biology Max-Planck-Institute for Biophysical Chemistry Am Fassberg 11, 37077 Göttingen (Germany) Fax:(+ 49)551-201-2202 E-mail: cigr@nmr.mpibpc.mpg.de Prof.Dr. S. Laufer Department of Pharmaceutical and Medicinal Chemistry Institute of Pharmacy, Eberhard-Karls-University Auf der Morgenstelle 8, 72076 Tübingen (Germany) [**] MAP: mitogen-activated protein. We thank Prof. Dr. Thilo Stehle, Eberhard-Karls-University, Tübingen, for the clone of p38a MAPK. This work was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft (grant no.: GRK1034), and the Fonds der Chemischen Industrie. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Zuschriften 3604 # 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. 2008, 120, 3604 –3607