Development of a high-throughput crystal structure-determination platform for JAK1 using a novel metal-chelator soaking system.
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.
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Nicole L
NL
Caspers
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Seungil
S
Han
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Francis
F
Rajamohan
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Lise R
LR
Hoth
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Kieran F
KF
Geoghegan
Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Timothy A
TA
Subashi
Inflammation Medicinal Chemistry, Pfizer Inc., 610 Main Street, Cambridge, MA 02139, USA.
Michael L
ML
Vazquez
Inflammation Medicinal Chemistry, Pfizer Inc., 610 Main Street, Cambridge, MA 02139, USA.
Neelu
N
Kaila
Oncology Structural Biology, Pfizer Inc., 10770 Science Center Drive, San Diego, CA 92121, USA.
Ciarán N
CN
Cronin
Oncology Structural Biology, Pfizer Inc., 10770 Science Center Drive, San Diego, CA 92121, USA.
Eric
E
Johnson
Structural Biology, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA.
Ravi G
RG
Kurumbail
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D004492:Edetic Acid / Q000737:chemistry*; D015870:Gene Expression; D006801:Humans; D053613:Janus Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
D002413:Cations, Divalent; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004492:Edetic Acid; C507904:JAK1 protein, human; D053613:Janus Kinase 1; D008274:Magnesium
Janus kinase; crystal soaking; kinases; ligand exchange; structure-based drug design