Structural analysis of an MK2–inhibitor complex: insight into the regulation of the secondary structure of the Gly-rich loop by TEI-I01800

Abstract

Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the complex of human MK2 (residues 41-364) with the potent MK2 inhibitor TEI-I01800 (pK(i) = 6.9) was determined at 2.9 A resolution. The MK2 structure in the MK2-TEI-I01800 complex is composed of two domains, as observed for other Ser/Thr kinases; however, the Gly-rich loop in the N-terminal domain forms an alpha-helix structure and not a beta-sheet. TEI-I01800 binds to the ATP-binding site as well as near the substrate-binding site of MK2. Both TEI-I01800 molecules have a nonplanar conformation that differs from those of other MK2 inhibitors deposited in the Protein Data Bank. The MK2-TEI-I01800 complex structure is the first active MK2 with an alpha-helical Gly-rich loop and TEI-I01800 regulates the secondary structure of the Gly-rich loop.

Structure of the β-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090

“…These long hydrogen bonds could be the reason why TEI-L03090 shows weak inhibition. Furthermore, the interactions between Cys140 and TEI-I01800 are important for MK2 activity, as described in our previous study (Fujino et al, 2010(Fujino et al, , 2013Kosugi et al, 2012). TEI-L03090 lacked van der Waals contacts with Cys140 because TEI-L03090 lacks a substituent at the 8-position corresponding to the p-ethyoxyphenyl group at the 7-position of TEI-I01800.…”

“…The human MK2 protein was purified using a modified version of our previously described method (Fujino et al, 2010). Purified MK2 was concentrated to 5 mg ml À1 (0.13 mM) and added to TEI-L03090 at a final concentration of 2 mM.…”

“…MK2 is a downstream kinase of p38 MAPK that is directly phosphorylated by p38 MAPK and that has also attracted attention as a target for anti-inflammatory therapy (Gaestel et al, 2007;Ronkina et al, 2010). In fact, several groups have developed programs for anti-inflammatory therapies by generating MK2 inhibitors and have reported crystal structures of MK2 (Wu et al, 2007;Hilling et al, 2007;Anderson et al, 2007;Revesz et al, 2010;Velcicky et al, 2010;Argiriadi et al, 2009Argiriadi et al, , 2010Fujino et al, 2010;Barf et al, 2011;Oubrie et al, 2012).…”

“…TEI-I01800 is highly potent and selective against significant kinases and has a 177-fold selectivity for MK2 over CDK2 (its IC 50 values for MK2 and CDK2 are 0.13 and 23 mM, respectively). Furthermore, we have previously reported crystal structures of MK2 and CDK2 in complex with TEI-I01800 (Fujino et al, 2010(Fujino et al, , 2013Kosugi et al, 2012), which revealed that MK2-TEI-I01800 forms an uncommon -helical glycine-rich loop ( -form) which is induced by the stable conformer of TEI-I01800. On the other hand, CDK2-TEI-I01800 adopted the -form commonly observed in CDK2 complexes with a small inhibitor.…”

“…This loop is part of a -hairpin that connects the 1 and 2 strands and forms a -sheet structure that interacts with ATP. MK2 also has a glycine-rich loop sequence 71 Gly-Leu-Gly-Ile-Asn-Gly 76 and, with the exception of two structures of complexes with inhibitors, TEI-I01800 (PDB entry 3a2c; Fujino et al, 2010) and a 2,4-diaminopyrimidine derivative from Abbott (PDB entry 3ka0; Argiriadi et al, 2010), all MK2 complexes deposited in the PDB have a -sheet glycine-rich loop ( -form).…”