A domain of all trades: The enzymatic versatility of the NiRAN domain.

The SARS-CoV-2 NiRAN domain is essential for viral replication. Despite adopting a pseudokinase fold, it catalyzes three distinct biochemical reactions from a single active site. In this issue of Molecular Cell, Small et al.1 elucidate the structural intricacies of the NiRAN domain shedding light on the factors that underlie its remarkable versatility.

Structural and mechanistic basis for protein glutamylation by the kinase fold.

The kinase domain transfers phosphate from ATP to substrates. However, the Legionella effector SidJ adopts a kinase fold, yet catalyzes calmodulin (CaM)-dependent glutamylation to inactivate the SidE ubiquitin ligases. The structural and mechanistic basis in which the kinase domain catalyzes protein glutamylation is unknown. Here we present cryo-EM reconstructions of SidJ:CaM:SidE reaction intermediate complexes. We show that the kinase-like active site of SidJ adenylates an active-site Glu in SidE, resulting in the formation of a stable reaction intermediate complex. An insertion in the catalytic loop of the kinase domain positions the donor Glu near the acyl-adenylate for peptide bond formation. Our structural analysis led us to discover that the SidJ paralog SdjA is a glutamylase that differentially regulates the SidE ligases during Legionella infection. Our results uncover the structural and mechanistic basis in which the kinase fold catalyzes non-ribosomal amino acid ligations and reveal an unappreciated level of SidE-family regulation.

The mechanism of RNA capping by SARS-CoV-2.

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

Redefining pseudokinases: A look at the untapped enzymatic potential of pseudokinases.

Catalytically inactive kinases, known as pseudokinases, are conserved in all three domains of life. Due to the lack of catalytic residues, pseudokinases are considered to act as allosteric regulators and scaffolding proteins with no enzymatic function. However, since these "dead" kinases are conserved along with their active counterparts, a role for pseudokinases may have been overlooked. In this review, we will discuss the recently characterized pseudokinases Selenoprotein O, Legionella effector SidJ, and the SARS-CoV2 protein nsp12 which catalyze AMPylation, glutamylation, and RNAylation, respectively. These studies provide structural and mechanistic insight into the versatility and diversity of the kinase fold.

A Legionella effector ADP-ribosyltransferase inactivates glutamate dehydrogenase.

ADP-ribosyltransferases (ARTs) are a widespread superfamily of enzymes frequently employed in pathogenic strategies of bacteria. Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaire's disease, has acquired over 330 translocated effectors that showcase remarkable biochemical and structural diversity. However, the ART effectors that influence L. pneumophila have not been well defined. Here, we took a bioinformatic approach to search the Legionella effector repertoire for additional divergent members of the ART superfamily and identified an ART domain in Legionella pneumophila gene0181, which we hereafter refer to as Legionella ADP-Ribosyltransferase 1 (Lart1) (Legionella ART 1). We show that L. pneumophila Lart1 targets a specific class of 120-kDa NAD+-dependent glutamate dehydrogenase (GDH) enzymes found in fungi and protists, including many natural hosts of Legionella. Lart1 targets a conserved arginine residue in the NAD+-binding pocket of GDH, thereby blocking oxidative deamination of glutamate. Therefore, Lart1 could be the first example of a Legionella effector which directly targets a host metabolic enzyme during infection.

The mechanism of RNA capping by SARS-CoV-2.

The SARS-CoV-2 RNA genome contains a 5'-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2'-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinase-like NiRAN domain5 of nsp12 transfers RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers RNA to GDP, forming the cap core structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N-terminus of nsp9 and the kinase-like active site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

Dynamic remodeling of host membranes by self-organizing bacterial effectors.

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

Bacterial pseudokinase catalyzes protein polyglutamylation to inhibit the SidE-family ubiquitin ligases.

Enzymes with a protein kinase fold transfer phosphate from adenosine 5'-triphosphate (ATP) to substrates in a process known as phosphorylation. Here, we show that the Legionella meta-effector SidJ adopts a protein kinase fold, yet unexpectedly catalyzes protein polyglutamylation. SidJ is activated by host-cell calmodulin to polyglutamylate the SidE family of ubiquitin (Ub) ligases. Crystal structures of the SidJ-calmodulin complex reveal a protein kinase fold that catalyzes ATP-dependent isopeptide bond formation between the amino group of free glutamate and the γ-carboxyl group of an active-site glutamate in SidE. We show that SidJ polyglutamylation of SidE, and the consequent inactivation of Ub ligase activity, is required for successful Legionella replication in a viable eukaryotic host cell.

SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK.

The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1-MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.