Reversible phosphorylation of cyclin T1 promotes assembly and stability of P-TEFb.

The positive transcription elongation factor b (P-TEFb) is a critical coactivator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC-mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

Recognition of a structural domain (RWDBD) in Gcn1 proteins that interacts with the RWD domain containing proteins.

The protein Gcn1 (General control non-derepressible 1) is found in virtually all eukaryotes, and is a key component of the general amino acid control signal transduction pathway. This pathway is best known for its importance for cells to sense and overcome amino acid starvation. Gcn1 directly binds to the RWD (RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases) domain of the protein kinase Gcn2, and this is essential for delivering the starvation signal to Gcn2. Gcn2, and thus the GAAC (General Amino Acid Control) pathway, then becomes activated enabling the cell to cope and overcome the starvation condition. Using sensitive homology detection and fold recognition methods a conserved structural domain in Gcn1, RWD Binding Domain (RWDBD), has been recognized that encompasses the region experimentally shown previously to be involved in Gcn2 binding. Further, the structural fold for this domain has been recognized as the ARM (Armadillo) domain, and residues likely to be involved in the binding of Gcn2 RWD domain have been identified within this structural domain. Thus, the current analysis provides a structural basis of Gcn1-Gcn2 association. REVIEWERS: This article was reviewed by Dr Oliviero Carugo and Dr Michael Gromiha.

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps.

Pre-mRNA splicing in eukaryotes is performed by the spliceosome, a highly complex macromolecular machine. SF3b is a multi-protein complex which recognizes the branch point adenosine of pre-mRNA as part of a larger U2 snRNP or U11/U12 di-snRNP in the dynamic spliceosome machinery. Although a cryo-EM map is available for human SF3b complex, the structure and relative spatial arrangement of all components in the complex are not yet known. We have recognized folds of domains in various proteins in the assembly and generated comparative models. Using an integrative approach involving structural and other experimental data, guided by the available cryo-EM density map, we deciphered a pseudo-atomic model of the closed form of SF3b which is found to be a "fuzzy complex" with highly flexible components and multiplicity of folds. Further, the model provides structural information for 5 proteins (SF3b10, SF3b155, SF3b145, SF3b130 and SF3b14b) and localization information for 4 proteins (SF3b10, SF3b145, SF3b130 and SF3b14b) in the assembly for the first time. Integration of this model with the available U11/U12 di-snRNP cryo-EM map enabled elucidation of an open form. This now provides new insights on the mechanistic features involved in the transition between closed and open forms pivoted by a hinge region in the SF3b155 protein that also harbors cancer causing mutations. Moreover, the open form guided model of the 5' end of U12 snRNA, which includes the branch point duplex, shows that the architecture of SF3b acts as a scaffold for U12 snRNA: pre-mRNA branch point duplex formation with potential implications for branch point adenosine recognition fidelity.

Use of evolutionary information in the fitting of atomic level protein models in low resolution cryo-EM map of a protein assembly improves the accuracy of the fitting.

Protein-protein interface residues, especially those at the core of the interface, exhibit higher conservation than residues in solvent exposed regions. Here, we explore the ability of this differential conservation to evaluate fittings of atomic models in low-resolution cryo-EM maps and select models from the ensemble of solutions that are often proposed by different model fitting techniques. As a prelude, using a non-redundant and high-resolution structural dataset involving 125 permanent and 95 transient complexes, we confirm that core interface residues are conserved significantly better than nearby non-interface residues and this result is used in the cryo-EM map analysis. From the analysis of inter-component interfaces in a set of fitted models associated with low-resolution cryo-EM maps of ribosomes, chaperones and proteasomes we note that a few poorly conserved residues occur at interfaces. Interestingly a few conserved residues are not in the interface, though they are close to the interface. These observations raise the potential requirement of refitting the models in the cryo-EM maps. We show that sampling an ensemble of models and selection of models with high residue conservation at the interface and in good agreement with the density helps in improving the accuracy of the fit. This study indicates that evolutionary information can serve as an additional input to improve and validate fitting of atomic models in cryo-EM density maps.

Improving the Accuracy of Fitted Atomic Models in Cryo-EM Density Maps of Protein Assemblies Using Evolutionary Information from Aligned Homologous Proteins.

Cryo-Electron Microscopy (cryo-EM) has become an important technique to obtain structural insights into large macromolecular assemblies. However the resolution of the density maps do not allow for its interpretation at atomic level. Hence they are combined with high resolution structures along with information from other experimental or bioinformatics techniques to obtain pseudo-atomic models. Here, we describe the use of evolutionary conservation of residues as obtained from protein structures and alignments of homologous proteins to detect errors in the fitting of atomic structures as well as improve accuracy of the protein-protein interfacial regions in the cryo-EM density maps.