title: Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.
abstract: Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Douglas
authors.initials: D
authors.lastname: Marcotte
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Mia
authors.initials: M
authors.lastname: Rushe
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Robert
authors.initials: R
authors.lastname: M Arduini
authors.affiliation: Beryllium Discovery Corp., 3 Preston Court, Bedford, MA, 01730.
authors.forename: Christine
authors.initials: C
authors.lastname: Lukacs
authors.affiliation: Beryllium Discovery Corp., 3 Preston Court, Bedford, MA, 01730.
authors.forename: Kateri
authors.initials: K
authors.lastname: Atkins
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Kevin
authors.initials: K
authors.lastname: Little
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Michael
authors.initials: M
authors.lastname: Cullivan
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Murugan
authors.initials: M
authors.lastname: Paramasivam
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Patterson
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Thomas
authors.initials: T
authors.lastname: Hesson
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Timothy
authors.initials: T
authors.lastname: D McKee
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Tricia L
authors.initials: TL
authors.lastname: May-Dracka
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Zhili
authors.initials: Z
authors.lastname: Xin
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Andrea
authors.initials: A
authors.lastname: Bertolotti-Ciarlet
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Govinda R
authors.initials: GR
authors.lastname: Bhisetti
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Lyssikatos
authors.affiliation: Department of Drug Discovery, Biogen Inc., 115 Binney Street, Cambridge, MA, 02142.
authors.forename: Laura F
authors.initials: LF
authors.lastname: Silvian
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D006801:Humans; D020125:Mutation, Missense; D000072417:Protein Domains; D047428:Protein Kinase Inhibitors / Q000737:chemistry*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary
chemical_list: D047428:Protein Kinase Inhibitors; C543044:MAP4K3 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: GCK-I; GLK; Map4k3; PEST sequence; activation loop; aminopyrrolopyrimidine inhibitor; crystal structure; germinal-center kinase-like kinase