TY - JOUR
T1 - Expression, purification and functional characterization of IκB kinase-2 (IKK-2) mutants
AU - Mathialagan, Sumathy
AU - Poda, Gennadiy I.
AU - Kurumbail, Ravi G.
AU - Selness, Shaun R.
AU - Hall, Troii
AU - Reitz, Beverly A.
AU - Weinberg, Robin A.
AU - Kishore, Nandini
AU - Mbalaviele, Gabriel
PY - 2010/8
Y1 - 2010/8
N2 - NF-κB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-κB 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.
AB - NF-κB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-κB 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.
KW - IKK-2
KW - NEMO
KW - NF-κB
KW - PHA-408
UR - http://www.scopus.com/inward/record.url?scp=77956202931&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2010.02.009
DO - 10.1016/j.pep.2010.02.009
M3 - Article
C2 - 20176108
AN - SCOPUS:77956202931
SN - 1046-5928
VL - 72
SP - 254
EP - 261
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -