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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.

SUBMITTER: Caspers NL

PROVIDER: S-EPMC5101585 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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

Caspers Nicole L NL Han Seungil S Rajamohan Francis F Hoth Lise R LR Geoghegan Kieran F KF Subashi Timothy A TA Vazquez Michael L ML Kaila Neelu N Cronin Ciarán N CN Johnson Eric E Kurumbail Ravi G RG

Acta crystallographica. Section F, Structural biology communications 20161027 Pt 11

Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg<sup>2+</sup>-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. ...[more]

PMID: 27827355

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