title: Expression, purification and functional characterization of IkappaB kinase-2 (IKK-2) mutants.
abstract: NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.
authors.affiliation: Pfizer Inc., 700 Chesterfield Parkway West, Chesterfield, MO 63017, USA.
authors.forename: Sumathy
authors.initials: S
authors.lastname: Mathialagan
authors.forename: Gennadiy I
authors.initials: GI
authors.lastname: Poda
authors.forename: Ravi G
authors.initials: RG
authors.lastname: Kurumbail
authors.forename: Shaun R
authors.initials: SR
authors.lastname: Selness
authors.forename: Troii
authors.initials: T
authors.lastname: Hall
authors.forename: Beverly A
authors.initials: BA
authors.lastname: Reitz
authors.forename: Robin A
authors.initials: RA
authors.lastname: Weinberg
authors.forename: Nandini
authors.initials: N
authors.lastname: Kishore
authors.forename: Gabriel
authors.initials: G
authors.lastname: Mbalaviele
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003198:Computer Simulation; D055808:Drug Discovery / Q000379:methods; D006801:Humans; D051550:I-kappa B Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007191:Indazoles / Q000737:chemistry / Q000378:metabolism; D007539:Isonicotinic Acids / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D040681:Structural Homology, Protein
chemical_list: D007191:Indazoles; D007539:Isonicotinic Acids; C548309:PHA 408; D011994:Recombinant Proteins; D051550:I-kappa B Kinase