Toso, D. B. | bioRxiv Science

bioRxiv2020

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 [A] resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.

biophysics↗

bioRxiv2020

The cryo-electron microscopy structure of the human CDK-activating kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II subunit Rpb1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell-cycle progression. Here, we have determined the three-dimensional structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and aid in the rational design of therapeutic compounds. SignificanceControl of gene expression and the cell cycle is critical for appropriate cell growth and timely cell division. Failure of the mechanisms regulating these processes can result in proliferative diseases. A molecular complex termed the CDK activating kinase (CAK) impinges on both of these regulatory networks in human cells and is thus a possible drug target for treatment of cancer. Here, we use cryo-electron microscopy to describe the detailed molecular structure of the human CAK, revealing its architecture and the interactions between its regulatory elements. Additionally, we have obtained the structure of the CAK in complex with a small-molecule inhibitor.

biochemistry↗