HD Exchange of p38 MAP Kinase

To determine how an inhibitor interacts with this target protein and changes its dynamics, H/D exchange of p38 MAP kinase was conducted with or without a small molecule inhibitor, SB203580 [55, 56]. The top row of Fig. 12.6 shows the H/D exchange pattern for unliganded p38 MAP kinase. p38 MAP kinase is organized about conformationally stable helices that include residues 74-86, 128-155, 203-213, 233-238, and 338-343. In contrast, residues 241-270 are especially mobile, even though they form a helix in the crystal structure [55, 57] (see also Fig. 12.7a).

A comparison of the exchange data for the unliganded (top row of Fig. 12.6) and liganded forms (middle row of Fig. 12.6) showed that in the SB203580 complex, two segments of p38 MAP kinase, residues 106-110 and 148-155, exhibited significant reductions in H/D exchange rates (bottom row of Fig. 12.6). The segments are separated in the amino acid sequence but adjacent in the folded struc-

P38 Map Kinase

Fig. 12.7 (a) Average deuteration level of each segment in apo p38 MAP kinase overlaid on the crystallographic structure (protein data bank ID: 1A9U) [56]. Blue indicates the region exchange slow and red the region exchange fast. (b) Ligand binding site identified by H/D exchange. Orange is the segment perturbed most. Dark blue is the regions that showed no H/D exchange perturbation. Light blue is SB203580. Gray indicates residues that were not analyzed [34].

Fig. 12.7 (a) Average deuteration level of each segment in apo p38 MAP kinase overlaid on the crystallographic structure (protein data bank ID: 1A9U) [56]. Blue indicates the region exchange slow and red the region exchange fast. (b) Ligand binding site identified by H/D exchange. Orange is the segment perturbed most. Dark blue is the regions that showed no H/D exchange perturbation. Light blue is SB203580. Gray indicates residues that were not analyzed [34].

ture, and encompass the ATP-binding residues and most of the activation loop (Fig. 12.7b). This kinase appears conformationally poised for this inhibitor, as no changes in dynamics were measured in other regions. The highly localized effects of SB203580 binding to p38 MAP kinase contrast with the global conformational changes induced by interactions with nucleotide substrates and protein partners that have been observed in other kinase systems [17, 22, 58].

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