Development of a high-throughput crystal structure-determination platform for JAK1 using a novel metal-chelator soaking system

Abstract

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg²⁺-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.

Keywords: Janus kinase; crystal soaking; kinases; ligand exchange; structure-based drug design.

Figure 1

Overall structure of the JAK1 kinase domain in complex with PF-­4950736.

Figure 2

Purity analysis of the JAK1 kinase domain. (a) 4–12% NuPAGE (Invitrogen) SDS denaturing gel. The left lane contains molecular-mass markers (labeled in kDa). (b) Size-exclusion chromatogram of the JAK1 kinase domain. The elution from SEC is later than expected owing to interaction of the protein with the stationary phase of the column.

Figure 3

Intact mass analysis of the crystallized form of the JAK1 kinase domain. The theoretical mass for the bisphosphorylated (abbreviation 2P) form of the protein was 36 366 Da. The mass discrepancy between this and the major observed mass (36 367 Da) was 27 p.p.m.

Figure 4

Crystal structure of the JAK1 kinase domain in complex with ADP and Mg²⁺ ion. ADP is shown in cyan and the (F _o − F _c) OMIT map is contoured at 2.5σ. Hydrogen bonds are depicted by red dashed lines.

Figure 5

Crystal structure of the JAK1 kinase domain in complex with PF-4950736. The compound is shown in magenta and the (F _o − F _c) OMIT map is contoured at 2.5σ. Hydrogen bonds are depicted by red dashed lines.