The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

and oncogenesis [1]. The common denominator of these processes is the initial C-terminal activation of ubiquitin The University of Alberta Edmonton, Alberta T6G 2H7 (Ub) by the activating enzyme (E1) followed by its subsequent transfer to a Ub-conjugating enzyme (E2) as a Canada 3 Department of Biochemistry and R.S. covalent E2-Ub thiolester intermediate. At this point, the mechanism of ubiquitination appears to diverge along McLaughlin Macromolecular Structure Facility either of two lines. In one case, Ub is transferred directly from the E2 to the lysine of a target protein in a reaction The University of Western Ontario London, Ontario N6A 5C1 that is facilitated by a Ub protein ligase (E3). In the other case, Ub is first transferred from the E2 to the active site Canada cysteine of an E3 as an E3-Ub thiolester intermediate, whereupon it is then transferred to the target protein. In either event, degradation of the target by the 26S Summary proteasome is facilitated by the assembly of a multi-Ub chain on the target in which the C terminus of each Ub Background: Ubiquitin-conjugating enzymes (E2s) are is linked to Lys48 (K48) of its neighbor. central enzymes involved in ubiquitin-mediated protein The ability of E2 proteins to orchestrate ubiquitination degradation. During this process, ubiquitin (Ub) and the through their interactions with Ub, E1, E3, and target E2 protein form an unstable E2-Ub thiolester intermediproteins makes them central players of the ubiquitin casate prior to the transfer of ubiquitin to an E3-ligase procade. All E2 proteins consist of a catalytic domain (150 tein and the labeling of a substrate for degradation. A residues) that includes the active site cysteine used to series of complex interactions occur among the target form the E2-Ub thiolester complex. Furthermore, X-ray substrate, ubiquitin, E2, and E3 in order to efficiently crystallographic structures of the catalytic domains from facilitate the transfer of the ubiquitin molecule. However, the E2 proteins Ubc1 (vide infra), Ubc2 [2], Ubc7 [3], and due to the inherent instability of the E2-Ub thiolester, Ubc9 [4] from Saccharomyces cerevisiae have shown the structural details of this complex intermediate are that this region is structurally conserved. Four ␣ helices not known. (␣1-␣4) essentially form one face of the protein, while a 4 strand antiparallel ␤ sheet (␤1-␤4) is found on the Results: A three-dimensional model of the E2-Ub thibackside of the enzyme between helices ␣1 and ␣2 in olester intermediate has been determined for the catathe sequence. The thiolester-forming cysteine is located lytic domain of the E2 protein Ubc1 (Ubc1 ⌬450 ) and ubion a relatively unstructured region (L2) ranging from 20 quitin from S. cerevisiae. The interface of the E2-Ub to 30 residues in length and linking ␤4 and ␣2. Recent intermediate was determined by kinetically monitoring X-ray crystallographic studies have shown that two thiolester formation by 1 H-15 N HSQC spectra by using structurally unrelated E3 proteins, E6AP and cCbl, intercombin...

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In 1 H 15 N HSQC ( 1 H 15 N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors.

A heterodimer composed of the catalytically active ubiquitin-conjugating enzyme hUbc13 and its catalytically inactive paralogue, hMms2, forms the catalytic core for the synthesis of an alternative type of multiubiquitin chain where ubiquitin molecules are tandemly linked to one another through a Lys-63 isopeptide bond. This type of linkage, as opposed to the more typical Lys-48-linked chains, serves as a non-proteolytic marker of protein targets involved in error-free postreplicative DNA repair and NF-B signal transduction. Using a two-dimensional 1 H-15 N NMR approach, we have mapped: 1) the interaction between the subunits of the human Ubc13⅐Mms2 heterodimer and 2) the interactions between each of the subunits or heterodimer with a non-covalently bound acceptor ubiquitin or a thiolesterlinked donor ubiquitin. Using these NMR-derived constraints and an unbiased docking approach, we have assembled the four components of this catalytic complex into a three-dimensional model that agrees well with its catalytic function.