Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

Many of the SARS-CoV-2 proteins have related counterparts across the Severe Acute Respiratory Syndrome (SARS-CoV) family. One such protein is non-structural protein 9 (Nsp9), which is thought to mediate viral replication, overall virulence, and viral genomic RNA reproduction. We sought to better characterize the SARS-CoV-2 Nsp9 and subsequently solved its X-ray crystal structure, in an apo form and, unexpectedly, in a peptide-bound form with a sequence originating from a rhinoviral 3C protease sequence (LEVL). The SARS-CoV-2 Nsp9 structure revealed the high level of structural conservation within the Nsp9 family. The exogenous peptide binding site is close to the dimer interface and impacted the relative juxtapositioning of the monomers within the homodimer. We have established a protocol for the production of SARS-CoV-2 Nsp9, determined its structure, and identified a peptide-binding site that warrants further study to understanding Nsp9 function

Inside-out: antibody-binding reveals potential folding hinge-points within the SARS-CoV-2 replication co-factor nsp9

Nsp9 is a conserved accessory component of the coronaviral replication and transcription complex. It is the predominant substrate of nsp12’s nucleotidylation activity while also serving to recruit proteins required for viral 5’-capping. Anti-nsp9 specific nanobodies have been isolated previously. We confirm that their binding mode is centred upon Trp-53 within SARS-CoV-2 nsp9. Antibody binding at this site surprisingly results in large-scale changes to the overall topology of this coronaviral unique fold. We further characterise the antibody-induced structural dynamism within nsp9, identifying a number of potentially flexible regions. A large expansion of the cavity between the s2-s3 and s4-s5 loops is particularly noteworthy. As is the potential for large-scale movements in the C-terminal GxxxG helix

Crystal structures of pertussis toxin with NAD(+) and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum, where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the endoplasmic reticulum into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (G alpha i) of heterotrimeric G proteins, thus promoting dysregulation of G protein-coupled receptor signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide and NAD+, which we name benzamide amino adenine dinucleotide. These crystal structures provide unprecedented insights into pre-and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small-molecule inhibitors

Crystal structures of pertussis toxin with NAD+ and analogs provide structural insights into the mechanism of its cytosolic ADP-ribosylation activity

Bordetella pertussis is the causative agent of whooping cough, a highly contagious respiratory disease. Pertussis toxin (PT), a major virulence factor secreted by B. pertussis, is an AB5-type protein complex topologically related to cholera toxin. The PT protein complex is internalized by host cells and follows a retrograde trafficking route to the endoplasmic reticulum (ER), where it subsequently dissociates. The released enzymatic S1 subunit is then translocated from the ER into the cytosol and subsequently ADP-ribosylates the inhibitory alpha-subunits (Gαi) of heterotrimeric G proteins, thus promoting dysregulation of G-protein coupled receptor (GPCR) signaling. However, the mechanistic details of the ADP-ribosylation activity of PT are not well understood. Here, we describe crystal structures of the S1 subunit in complex with nicotinamide adenine dinucleotide (NAD+), with NAD+ hydrolysis products ADP-ribose and nicotinamide, with NAD+ analog PJ34, and with a novel NAD+ analog formed upon S1 subunit crystallization with 3-amino benzamide (3AB) and NAD+, which we name benzamide amino adenine dinucleotide (BaAD). These crystal structures provide unprecedented insights into pre- and post-NAD+ hydrolysis steps of the ADP-ribosyltransferase activity of PT. We propose that these data may aid in rational drug design approaches and further development of PT-specific small molecule inhibitors

Cloning, expression, purification and crystallographic studies of galectin-11 from domestic sheep (Ovis aries)

Galectins are an evolutionarily conserved family of proteins that translate glycan recognition into cellular effects. Galectin-11 is a unique member of the galectin family that is only expressed in ruminants such as sheep, goat and cattle and that plays a critical role in several important biological processes, such as reproduction and parasite-mediated innate immune responses. Currently, these two areas are of major importance for the sustainability of ruminant livestock production. Despite the emerging biological significance of galectin-11, no structural information is available. It is expected that structural studies will unravel the functional mechanisms of galectin-11 activity. Here, the expression, purification and crystallization of the ruminant-specific galectin-11 from domestic sheep and the collection of X-ray data to 2.0 Å resolution are reported. © 2015

Disrupting the allosteric interaction between the plasmodium falciparumcAMP-dependent kinase and its regulatory subunit

The ubiquitous second messenger cAMP mediates signal transduction processes in the malarial parasite that regulate host erythrocyte invasion and the proliferation of merozoites. In Plasmodium falciparum, the central receptor for cAMP is the single regulatory subunit (R) of protein kinase A (PKA). To aid the development of compounds that can selectively dysregulate parasite PKA signaling, we solved the structure of the PKA regulatory subunit in complex with cAMP and a related analogue that displays antimalarial activity, (Sp)-2-Cl-cAMPS. Prior to signaling, PKA-R holds the kinase's catalytic subunit (C) in an inactive state by exerting an allosteric inhibitory effect. When two cAMP molecules bind to PKA-R, they stabilize a structural conformation that facilitates its dissociation, freeing PKA-C to phosphorylate downstream substrates such as apical membrane antigen 1. Although PKA activity was known to be necessary for erythrocytic proliferation, we show that uncontrolled induction of PKA activity using membrane-permeable agonists is equally disruptive to growth

FusC, a member of the M16 protease family acquired by bacteria for iron piracy against plants.

Iron is essential for life. Accessing iron from the environment can be a limiting factor that determines success in a given environmental niche. For bacteria, access of chelated iron from the environment is often mediated by TonB-dependent transporters (TBDTs), which are β-barrel proteins that form sophisticated channels in the outer membrane. Reports of iron-bearing proteins being used as a source of iron indicate specific protein import reactions across the bacterial outer membrane. The molecular mechanism by which a folded protein can be imported in this way had remained mysterious, as did the evolutionary process that could lead to such a protein import pathway. How does the bacterium evolve the specificity factors that would be required to select and import a protein encoded on another organism's genome? We describe here a model whereby the plant iron-bearing protein ferredoxin can be imported across the outer membrane of the plant pathogen Pectobacterium by means of a Brownian ratchet mechanism, thereby liberating iron into the bacterium to enable its growth in plant tissues. This import pathway is facilitated by FusC, a member of the same protein family as the mitochondrial processing peptidase (MPP). The Brownian ratchet depends on binding sites discovered in crystal structures of FusC that engage a linear segment of the plant protein ferredoxin. Sequence relationships suggest that the bacterial gene encoding FusC has previously unappreciated homologues in plants and that the protein import mechanism employed by the bacterium is an evolutionary echo of the protein import pathway in plant mitochondria and plastids

Complimentary electrostatics dominate T-cell receptor binding to a psoriasis-associated peptide antigen presented by human leukocyte antigen C∗06:02

Psoriasis is a chronic skin disease characterized by hyperproliferative epidermal lesions infiltrated by autoreactive T cells. Individuals expressing the human leukocyte antigen (HLA) C∗06:02 allele are at highest risk for developing psoriasis. An autoreactive T cell clone (termed Vα3S1/Vβ13S1) isolated from psoriatic plaques is selective for HLA-C∗06:02, presenting a peptide derived from the melanocyte-specific autoantigen ADAMTSL5 (VRSRRCLRL). Here we determine the crystal structure of this psoriatic TCR–HLA-C∗06:02 ADAMTSL5 complex with a stabilized peptide. Docking of the TCR involves an extensive complementary charge network formed between negatively charged TCR residues interleaving with exposed arginine residues from the self-peptide and the HLA-C∗06:02 α1 helix. We probed these interactions through mutagenesis and activation assays. The charged interface spans the polymorphic region of the C1/C2 HLA group. Notably the peptide-binding groove of HLA-C∗06:02 appears exquisitely suited for presenting highly charged Arg-rich epitopes recognized by this acidic psoriatic TCR. Overall, we provide a structural basis for understanding the engagement of melanocyte antigen-presenting cells by a TCR implicated in psoriasis while simultaneously expanding our knowledge of how TCRs engage HLA-C

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition.

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity

A natural product compound inhibits coronaviral replication in vitro by binding to the conserved Nsp9 SARS-CoV-2 protein

The Nsp9 replicase is a conserved coronaviral protein that acts as an essential accessory component of the multi-subunit viral replication/transcription complex. Nsp9 is the predominant substrate for the essential nucleotidylation activity of Nsp12. Compounds specifically interfering with this viral activity would facilitate its study. Using a native mass-spectrometry-based approach to screen a natural product library for Nsp9 binders, we identified an ent-kaurane natural product, oridonin, capable of binding to purified SARS-CoV-2 Nsp9 with micromolar affinities. By determining the crystal structure of the Nsp9-oridonin complex, we showed that oridonin binds through a conserved site near Nsp9’s C-terminal GxxxG-helix. In enzymatic assays, oridonin’s binding to Nsp9 reduces its potential to act as substrate for Nsp12’s Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain. We also showed using in vitro cellular assays oridonin, while cytotoxic at higher doses has broad antiviral activity, reducing viral titer following infection with either SARS-CoV-2 or, to a lesser extent, MERS-CoV. Accordingly, these preliminary findings suggest that the oridonin molecular scaffold may have the potential to be developed into an antiviral compound to inhibit the function of Nsp9 during coronaviral replication