ABSTRACT
Human checkpoint kinase ataxia telangiectasia-mutated (ATM) plays a key role in initiation of the DNA damage response following DNA double-strand breaks. ATM inhibition is a promising approach in cancer therapy, but, so far, detailed insights into the binding modes of known ATM inhibitors have been hampered due to the lack of high-resolution ATM structures. Using cryo-EM, we have determined the structure of human ATM to an overall resolution sufficient to build a near-complete atomic model and identify two hitherto unknown zinc-binding motifs. We determined the structure of the kinase domain bound to ATPγS and to the ATM inhibitors KU-55933 and M4076 at 2.8 Å, 2.8 Å and 3.0 Å resolution, respectively. The mode of action and selectivity of the ATM inhibitors can be explained by structural comparison and provide a framework for structure-based drug design.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Ataxia Telangiectasia Mutated Proteins/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Binding Sites , Catalytic Domain , Cryoelectron Microscopy , Humans , Models, Molecular , Morpholines/chemistry , Morpholines/metabolism , Mutation , Neoplasms/genetics , Protein Conformation , Pyrones/chemistry , Pyrones/metabolismABSTRACT
The central component AcrB of the Escherichia coli drug efflux complex AcrA-AcrB-TolC has been extensively investigated by X-ray crystallography of detergent-protein 3-D crystals. In these crystals, AcrB packs as trimers - the functional unit. We visualized the AcrB-AcrB interaction in its native environment by examining E. coli lipid reconstituted 2-D crystals, which were overwhelmingly formed by asymmetric trimers stabilized by strongly-interacting monomers from adjacent trimers. Most interestingly, we observed lattices formed by an arrangement of AcrB monomers distinct from that in traditional trimers. This hitherto unobserved packing, might play a role in the biogenesis of trimeric AcrB.
