PDBsum entry 3f3t

Transferase

PDB id: 3f3t

Contents

Protein chains

261 a.a.

Ligands

1AU ×2

Waters ×98

References listed in PDB file

Key reference

Title

A new screening assay for allosteric inhibitors of csrc.

Authors

J.R.Simard, S.Klüter, C.Grütter, M.Getlik, M.Rabiller, H.B.Rode, D.Rauh.

Ref.

Nat Chem Biol, 2009, 5, 394-396. [DOI no: 10.1038/nchembio.162]

PubMed id

19396179

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

Abstract

Targeting kinases outside the highly conserved ATP pocket is thought to be a promising strategy for overcoming bottlenecks in kinase inhibitor research, such as limited selectivity and drug resistance. Here we report the development and application of a direct binding assay to detect small molecules that stabilize the inactive conformation of the tyrosine kinase cSrc. Protein X-ray crystallography validated the assay results and confirmed an exclusively allosteric binding mode.

Figure 1.
(a) Structural model of a protein kinase of interest with the DFG motif (orange) and activation loop (red) highlighted. A cysteine was mutated into the activation loop of cSrc for subsequent labeling with the environmentally sensitive fluorophore acrylodan to generate a sensitive DFG-out fluorescence-based binding assay. The DFG-out conformation is stabilized by the binding of allosteric type III inhibitors (blue surface representation) or type II inhibitors that lock the kinase in the inactive state. The binding of ATP or type I inhibitors (yellow surface representation) stabilizes the active DFG-in conformation. Both conformations are in equilibrium and result from structural changes in the activation loop and the DFG motif. (b) Examples of type I, type II and type III kinase inhibitors and scaffolds. (c) In the absence of ligand, acrylodan-labeled cSrc shows two emission maxima at 475 nm and 505 nm. Type I ligands induce a robust loss of fluorescence intensity (represented by red arrows) at 475 nm, resulting in a red shift in the emission maxima to 510 nm (right panel). Type II and III inhibitors stabilize the inactive kinase conformation and elicit a different response in which the emissions at 475 nm and 505 nm are equally reduced (left panel). The emission signal at 445 nm is less sensitive to ligand binding and serves as an internal reference point, allowing for more stable ratiometric fluorescence measurements and K[d] determinations.

Figure 3.
(a) cSrc in complex with the type III inhibitor 1b. (b) cSrc in complex with the type III inhibitor 1c. Electron density maps (2F[o] – F[c]) of cSrc (gray) and 1b, 1c (red) are contoured at 1 . Hydrogen bonding interactions of the inhibitor with helix C (blue) and the backbone of the DFG motif (orange) stabilize the inhibitor in the allosteric site and are highlighted by dotted lines (red). The hinge region (pink) of the kinase domain (represented by Met341) is not contacted by either inhibitor. (c) Alignment of the cSrc–1c complex with BIRB-796 bound to p38 (yellow) (Protein Data Bank code 1KV2). Hydrogen bonds of the p38 –BIRB-796 complex are highlighted by dotted lines (red). The pyrazolourea moiety of both ligands resides in the allosteric site, and the naphthyl side chain is located in the hydrophobic subpocket close to the gatekeeper residue (Thr106 in p38 and Thr338 in cSrc). The morpholino group of BIRB-796 binds to the hinge region of p38 . Although the binding mode of the aryl pyrazolourea scaffold is conserved in both complexes, BIRB-796 binds only weakly to cSrc with a K[d] > 10 M. Coordinates and structure factors have been deposited under the following Protein Data Bank accession codes: cSrc bound to 1b, 3F3U; cSrc bound to 1c, 3F3T.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Chem Biol (2009, 5, 394-396) copyright 2009.