Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

Metazoan retromer (VPS26/VPS35/VPS29) associates with sorting nexins on endosomal tubules to sort proteins to the trans-Golgi network or plasma membrane. Mechanisms of metazoan retromer assembly remain undefined. We combine single-particle cryoelectron microscopy with biophysical methods to uncover multiple oligomer structures. 2D class averages reveal mammalian heterotrimers; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical solution studies. We provide reconstructions of all species, including key sub-structures (~5 Å resolution). Local resolution variation suggests that heterotrimers and dimers adopt multiple conformations. Our structures identify a flexible, highly conserved electrostatic dimeric interface formed by VPS35 subunits. We generate structure-based mutants to disrupt this interface in vitro. Equivalent mutations in yeast demonstrate a mild cargo-sorting defect. Our data suggest the metazoan retromer is an adaptable and plastic scaffold that accommodates interactions with different sorting nexins to sort multiple cargoes from endosomes their final destinations.

The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway.

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here, we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA, and will aid in the development of new anti-viral therapeutics.

The adaptor protein 4 (AP4) complex (ε/β4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network (TGN). AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegia. We have used a range of structural, biochemical, and biophysical approaches to determine the molecular basis for how the AP4 β4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal β4 appendage domain. Using NMR chemical shift mapping, we define the binding site on β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish binding in vitro. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4 coated vesicles in cell biology, and especially in neuronal function.

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is a uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3′ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3′ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4) coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin ENTH and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8, and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1,500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.