ABSTRACT
We report the 3.45-Å resolution cryo-EM structure of human SMG1-SMG8-SMG9, a phosphatidylinositol-3-kinase (PI(3)K)-related protein kinase (PIKK) complex central to messenger RNA surveillance. Structural and MS analyses reveal the presence of inositol hexaphosphate (InsP6) in the SMG1 kinase. We show that the InsP6-binding site is conserved in mammalian target of rapamycin (mTOR) and potentially other PIKK members, and that it is required for optimal in vitro phosphorylation of both SMG1 and mTOR substrates.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Phytic Acid/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Binding Sites , Cryoelectron Microscopy , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Models, Molecular , Phytic Acid/chemistry , Protein Binding , Protein Conformation , Protein Kinases/chemistry , Protein Kinases/ultrastructure , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/ultrastructure , RNA StabilityABSTRACT
TMC-95's natural cyclic tripeptide metabolites represent potent competitive proteasome inhibitors. The constrained conformation of TMC-95 proteasomal inhibitors provides the driving force for entropically high-affinity binding. Based on the crystal structure of the proteasome:TMC-95A complex, the synthetically challenging TMC-95 core structure was used for the design and synthesis of less demanding biphenyl-ether macrocycles, in which the biphenyl-ether moiety functions as an endocyclic clamp restricting its tripeptide backbone. These simplified analogs allowed us to identify high plasticity of the proteasomal tryptic-like specificity pocket. Biphenyl-ether compounds extended with an amide group were hydrolyzed by the proteasome, although the crystal structure of such proteasome:biphenyl-ether complexes revealed quenching of proteolysis at the acyl-enzyme intermediate. Our data reveal that biphenyl-ether derivatives bind noncovalently to the proteasomal tryptic-like active site in a reversible substrate-like manner without allosteric changes of active site residues.
Subject(s)
Drug Design , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Binding Sites , Catalysis , Cross-Linking Reagents/chemistry , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Peptides, Cyclic/chemical synthesis , Phenyl Ethers/chemistry , Protease Inhibitors/chemistry , Proteasome Endopeptidase Complex/chemistry , Saccharomyces cerevisiae/enzymology , Structure-Activity Relationship , Substrate Specificity , Trypsin/metabolism , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/chemistry , Trypsin Inhibitors/pharmacologyABSTRACT
The complex thermodynamics that govern noncovalent protein-ligand interactions are still not fully understood, despite the exponential increase in experimental structural data available from X-ray crystallography and NMR spectroscopy. The eukaryotic 20S proteasome offers an ideal system for such studies as it contains in duplicate three proteolytically active sites with different substrate specificities. The natural product TMC-95A inhibits these proteolytic centers noncovalently with distinct affinities. X-ray crystallographic analysis of the complexes of the yeast proteasome core particle with this natural inhibitor and two synthetic analogues clearly revealed highly homologous hydrogen-bonding networks involving mainly the peptide backbone despite the strongly differentiated binding affinities to the three active sites of the 20S proteasome. The natural product and the two analogues are constrained in a rigid beta-type extended conformation by the endocyclic biaryl clamp, which preorganizes the peptide backbone for optimal adaptation of the ligands to the active site clefts and thus favors the binding processes entropically. However, the biaryl clamp also dictates the orientation of the P1 and P3 residues and their mode of interaction with the protein binding subsites. This limitation is optimally solved in TMC-95A with the conformationally restricted (Z)-prop-1-enyl group acting as P1 residue, at least for the chymotrypsin-like active site; however, it critically affects the inhibitory potencies of the analogues, thus suggesting the use of less-rigid endocyclic clamps in the design of proteasome inhibitors that allow for a better presentation of residues interacting with the active site clefts of the enzyme.
