High-resolution cryo-EM of the human CDK-activating kinase for structure-based drug design.

Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.

Cryo-EM structure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.