ABSTRACT
We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.
Subject(s)
Chemistry, Pharmaceutical/methods , Combinatorial Chemistry Techniques/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinases , Binding Sites/drug effects , Crystallography, X-Ray , Drug Design , Ligands , Mass Spectrometry/methods , Models, Chemical , Molecular Structure , Purines/chemistry , Structure-Activity RelationshipABSTRACT
The family of calcium binding proteins called KChIPs associates with Kv4 family K(+) channels and modulates their biophysical properties. Here, using mutagenesis and X-ray crystallography, we explore the interaction between Kv4 subunits and KChIP1. Two regions in the Kv4.2 N terminus, residues 7-11 and 71-90, are necessary for KChIP1 modulation and interaction with Kv4.2. When inserted into the Kv1.2 N terminus, residues 71-90 of Kv4.2 are also sufficient to confer association with KChIP1. To provide a structural framework for these data, we solved the crystal structures of Kv4.3N and KChIP1 individually. Taken together with the mutagenesis data, the individual structures suggest that that the Kv4 N terminus is required for stable association with KChIP1, perhaps through a hydrophobic surface interaction, and that residues 71-90 in Kv4 subunits form a contact loop that mediates the specific association of KChIPs with Kv4 subunits.
Subject(s)
Calcium-Binding Proteins/chemistry , Cell Membrane/chemistry , Potassium Channels, Voltage-Gated , Potassium Channels/chemistry , Amino Acid Sequence/physiology , Animals , Binding Sites/genetics , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Crystallography, X-Ray , Humans , Kv Channel-Interacting Proteins , Membrane Potentials/genetics , Models, Molecular , Mutagenesis, Site-Directed/genetics , Oocytes/metabolism , Patch-Clamp Techniques , Potassium Channels/genetics , Potassium Channels/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Protein Subunits , Shal Potassium ChannelsABSTRACT
MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.