In eukaryotic cells, apoptosis and cell cycle arrest by the Ras 3 RASSF 3 MST pathway are controlled by the interaction of SARAH (for Salvador/Rassf/Hippo) domains in the C-terminal part of tumor suppressor proteins. The Mst1 SARAH domain interacts with its homologous domain of Rassf1 and Rassf5 (also known as Nore1) by forming a heterodimer that mediates the apoptosis process. Here, we describe the homodimeric structure of the human Mst1 SARAH domain and its heterotypic interaction with the Rassf5 and Salvador (Sav) SARAH domain. The Mst1 SARAH structure forms a homodimer containing two helices per monomer. An antiparallel arrangement of the long ␣-helices (h2/h2 ) provides an elongated binding interface between the two monomers, and the short 3 10 helices (h1/h1 ) are folded toward that of the other monomer. Chemical shift perturbation experiments identified an elongated, tight-binding interface with the Rassf5 SARAH domain and a 1:1 heterodimer formation. The linker region between the kinase and the SARAH domain is shown to be disordered in the free protein. These results imply a novel mode of interaction with RASSF family proteins and provide insight into the mechanism of apoptosis control by the SARAH domain.tumor suppressor ͉ cell cycle arrest ͉ Hippo ͉ Salvador R ecent work in cellular homeostasis has uncovered a pathway mediated by the MST (mammalian sterile 20-like kinase) family, the human ortholog for Hippo (Hpo), which promotes apoptosis and restricts cell proliferation in conjunction with RASSF family tumor suppressors and/or the scaffold protein Salvador (Sav) (1-6). This pathway is characterized by a unique interaction motif called SARAH (for Sav/Rassf/Hpo), which connects the proteins involved in this pathway (7).Mammalian sterile 20-like kinase 1 (Mst1, also called STK4) is a member of a family of serine/threonine kinases that show similarity to Ste20, an upstream activator of the MAPK pathway in budding yeast (8,9). Mst1 is cleaved by caspase 3, which is triggered either by the activation of death receptors, such as Fas and the TNF-␣ receptor, or by exposure of the cells to inducers of apoptosis, such as staurosporine or ceramide (10-13). Whereas intact Mst1 is localized predominantly in the cytoplasm, the catalytic fragment of Mst1 generated by caspase-mediated cleavage translocates to the nucleus and phosphorylates histone H2B at Ser-14, resulting in chromatin condensation, DNA fragmentation, and, ultimately, cell death by apoptosis (14,15).A Drosophila homolog of Mst1/2, Hippo (Hpo), together with Salvador (Sav) and Warts (Wts), promotes both proper exit from the cell cycle and apoptosis during development (1-3). Mst1 and Mst2 have also been shown to associate with members of the RASSF family of tumor suppressors, such as Rassf1 and Rassf5 (also known as Nore1), all of which contain a conserved Rasassociation (RA) domain, with both of the MST and RASSF proteins colocalizing to microtubules throughout the cell cycle (4-6). Whereas purified recombinant Rassf1A inhibited the kinase activity of ...

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 Å) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.backbone NMR structure | cell-free synthesis | combinatorial selective labeling

The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX–TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation.

Herpesvirus-associated ubiquitin specific protease (HAUSP) regulates the stability of p53 and MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated anti-tumor activity. Here, we report the structural analysis of HAUSP with Kaposi’s sarcoma-associated herpesvirus vIRF4 and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction resulting in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, while the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Consequently, peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell cycle arrest and apoptosis in culture and tumor regression in xenograft mouse model. Thus, the virus has developed a unique molecular strategy to target the HAUSP-MDM2-p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists.

The heterodimeric structure of the MST1 and RASSF5 SARAH domains is presented. A comparison of homodimeric and heterodimeric interactions provides a structural basis for the preferential association of the SARAH heterodimer.

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titres of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 3' end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3' end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method.