Structural Basis for a High Affinity Inhibitor Bound to Protein Kinase MK2

Abstract

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.

Introduction

Protein kinases have become major drug targets in the pharmaceutical industry, and a number of low molecular mass inhibitors are in the clinic now (for reviews, see Noble et al.¹ and Schaefer & Egner²). Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAP-kinase 2, or MK2) is a serine/threonine kinase involved in inflammation. It is considered a molecular target for therapeutic intervention in indications such as inflammation and cancer (for reviews see Kotlyarov & Gaestel³ and Gaestel,⁴ and references therein). MK2 is a member of the p38 MAP kinase pathway, which mediates response to stress and is activated by heatshock, UV, lipopolysaccharides (LPS), and pro-inflammatory cytokines.⁵ An MK2 knock-out study in mice revealed a reduction in TNF-α, interferon-γ, IL-1β, and IL-6 and an increased survival upon challenge with LPS, suggesting that MK2 is a key regulator of the inflammatory process.⁶ Recently, MK2 has also been identified as a cell cycle checkpoint kinase playing a role in DNA damage response.⁷

Human MK2 features 400 residues with an N-terminal proline-rich region (residues 10–44), a kinase domain (residues 51–325), and a C-terminal autoinhibitory region (residues 328–364). The latter region contains a nuclear export signal of hydrophobic residues (356–368) while the far C terminus features a bipartite nuclear localization signal of basic residues (373–389).8., 9., 10., 11., 12., 13. Previously, crystal structures of MK2 in the apo form¹⁴ and bound to AMPPNP,¹⁵ ADP and the broad kinase inhibitor staurosporine¹⁶ have been determined. Apo MK2 featured an inactive kinase conformation with an unusually wide open ATP pocket. In addition, the substrate-binding site was blocked by the C-terminal autoinhibitory region, which formed a long helix and mimicked substrate binding.¹⁴ The very open ATP pocket did not appear favorable for high affinity binding of inhibitors. The structures with ADP and staurosporine then revealed the active confirmation of MK2 and showed that here, the ATP site adopted a more closed conformation.¹⁶ However, these ligands bind only with low affinity with IC50 values in the micromolar range. In contrast, compound-1 (Figure 1) is a high affinity MK2 inhibitor (IC50 8.5 nM).¹⁷ In order to understand the ATP pocket of MK2 and the molecular basis for high affinity interaction with this binding site, we determined the crystal structure of MK2 in complex with compound-1. We report two structures, produced from soaking into pre-formed crystals of MK2:ADP and from co-crystallization. They will help in the design of further potent and selective inhibitors of MK2 for the treatment of inflammatory diseases and cancer.