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

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

Applications of ¹⁹F-NMR in fragment-based drug discovery

19F-NMR has proved to be a valuable tool in fragment-based drug discovery. Its applications include screening libraries of fluorinated fragments, assessing competition among elaborated fragments and identifying the binding poses of promising hits. By observing fluorine in both the ligand and the target protein, useful information can be obtained on not only the binding pose but also the dynamics of ligand-protein interactions. These applications of 19F-NMR will be illustrated in this review with studies from our fragment-based drug discovery campaigns against protein targets in parasitic and infectious diseases

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright (C) 2016 John Wiley & Sons, Ltd

Structure and Functional Characterization of the Conserved JAK Interaction Region in the Intrinsically Disordered N‑Terminus of SOCS5

SOCS5 can negatively regulate both JAK/STAT and EGF-receptor pathways and has therefore been implicated in regulating both the immune response and tumorigenesis. Understanding the molecular basis for SOCS5 activity may reveal novel ways to target key components of these signaling pathways. The N-terminal region of SOCS5 coordinates critical protein interactions involved in inhibition of JAK/STAT signaling, and a conserved region within the N-terminus of SOCS5 mediates direct binding to the JAK kinase domain. Here we have characterized the solution conformation of this conserved JAK interaction region (JIR) within the largely disordered N-terminus of SOCS5. Using nuclear magnetic resonance (NMR) chemical shift analysis, relaxation measurements, and NOE analysis, we demonstrate the presence of preformed structural elements in the JIR of mouse SOCS5 (mSOCS5_175–244), consisting of an α-helix encompassing residues 224–233, preceded by a turn and an extended structure. We have identified a phosphorylation site (Ser211) within the JIR of mSOCS5 and have investigated the role of phosphorylation in modulating JAK binding using site-directed mutagenesis

SOCS5-SH2 domain binding analysis and identification of Shc-1 pY317 as a high affinity-potential binding target.

<p>SPR analysis of phosphopeptide binding to the SOCS5-SH2 domain. A constant amount of recombinant SOCS5 was mixed with serially diluted phosphopeptides (0.4–10 µM) and flowed over immobilised Shc-1 pY317 peptide. The response units are expressed as a percentage of maximal binding in the absence of competitor and are plotted against the concentration of competitor peptide. Steady-state analysis at saturation of binding was used to derive the <i>K</i>_D values for the respective phosphopeptides. Binding analysis of (<b>A</b>) JAK, Shc-1, or wild-type and (<b>B</b>) mutated EGF-R phosphopeptides. Phosphopeptide sequences and the respective <i>K</i>_D values are shown in the right-hand side table. Yellow boxes highlight residues replaced by an alanine residue. (<b>C</b>) Structural model of the SOCS5-SH2-Shc-1 peptide complex. A homology model for the SOCS5-SH2 domain was built using the SOCS4 crystal structure as a template (PDB code 2IZV). The Shc-1 pY317 peptide was modelled from the SOCS3-gp130 crystal structure (PDB code 2HMH). Side chains were optimized using ICM-PRO (Molsoft). The backbone of the flexible EF and BG loops was fixed in the apo-SOCS4 conformation, but is likely to adjust on peptide binding to maximize interactions. Predicted hydrogen bonds are shown as dashed lines. (<b>D</b>) SOCS5 interacts with full-length Shc-1 protein. 293T cells were transfected with cDNA encoding Myc-tagged SOCS5 (+) in the presence (+) or absence of cDNA encoding Flag-tagged Shc-1 or alternatively, with cDNA encoding Flag-tagged SOCS5 alone. Cells were treated with 10 μM MG132 for 3.5 h prior to treatment with sodium pervanadate solution for 30 min. Cells were then lysed and anti-Flag immunoprecipitates analyzed by Western blot with anti-SOCS5 antibodies (αSOCS5). The blots were stripped and reprobed with a phospho-specific antibody for Shc-1-Y317 (middle panel). Cell lysates were analyzed by Western blot with anti-SOCS5 (lower panel).</p

An N-terminal fragment corresponding to residues 175–244 of SOCS5 can directly bind JAK1.

<p>(A) SPR analysis of SOCS5^175–244 fragment binding to the JAK JH1 domain. Serially diluted JAK JH1 domains (62.5 nM–2 μM) were flowed over immobilised SOCS5^175–244 protein. Upper panels represent sensorgrams showing the kinetics of binding. Lower panels show steady-state analysis. (B) 293T cells were transfected with the Stat6 reporter and increasing amounts of cDNA expressing Flag-tagged SOCS5 (3.13–100 ng) or SOCS5 lacking the conserved N-terminal fragment (9.5–300 ng; Δ175–244) and stimulated overnight with 10 ng/mL rhIL-4. Cells were lysed and induced luciferase activity measured and normalised according to Renilla activity. Data are expressed as arbitrary units and represent the mean of triplicates ± SD. Cell lysates were analyzed by Western blotting for Flag-tagged proteins (SOCS5 upper; Δ175–244 lower panel); images were generated from the same gel and exposure. (C) Recombinant SOCS5 JIR or SOCS3 was incubated with 20 nM JAK1 and GST-JAK2 activation peptide (substrate; GST-J) for 15 min in the presence of 2.5 mM Mg/³²P-γ-ATP at 37°C. Incorporation of ³²P was visualised by autoradiography (top panel) and protein input by SDS-PAGE and Coomassie staining (lower panel).</p

Suppressor of Cytokine Signaling (SOCS) 5 Utilises Distinct Domains for Regulation of JAK1 and Interaction with the Adaptor Protein Shc-1

<div><p>Suppressor of Cytokine Signaling (SOCS)5 is thought to act as a tumour suppressor through negative regulation of JAK/STAT and epidermal growth factor (EGF) signaling. However, the mechanism/s by which SOCS5 acts on these two distinct pathways is unclear. We show for the first time that SOCS5 can interact directly with JAK via a unique, conserved region in its N-terminus, which we have termed the JAK interaction region (JIR). Co-expression of SOCS5 was able to specifically reduce JAK1 and JAK2 (but not JAK3 or TYK2) autophosphorylation and this function required both the conserved JIR and additional sequences within the long SOCS5 N-terminal region. We further demonstrate that SOCS5 can directly inhibit JAK1 kinase activity, although its mechanism of action appears distinct from that of SOCS1 and SOCS3. In addition, we identify phosphoTyr317 in Shc-1 as a high-affinity substrate for the SOCS5-SH2 domain and suggest that SOCS5 may negatively regulate EGF and growth factor-driven Shc-1 signaling by binding to this site. These findings suggest that different domains in SOCS5 contribute to two distinct mechanisms for regulation of cytokine and growth factor signaling.</p></div

Structure and Dynamics of Apical Membrane Antigen 1 from <i>Plasmodium falciparum</i> FVO

Apical membrane antigen 1 (AMA1) interacts with RON2 to form a protein complex that plays a key role in the invasion of host cells by malaria parasites. Blocking this protein–protein interaction represents a potential route to controlling malaria and related parasitic diseases, but the polymorphic nature of AMA1 has proven to be a major challenge to vaccine-induced antibodies and peptide inhibitors exerting strain-transcending inhibitory effects. Here we present the X-ray crystal structure of AMA1 domains I and II from <i>Plasmodium falciparum</i> strain FVO. We compare our new structure to those of AMA1 from <i>P. falciparum</i> 3D7 and <i>Plasmodium vivax</i>. A combination of normalized <i>B</i> factor analysis and computational methods has been used to investigate the flexibility of the domain I loops and how this correlates with their roles in determining the strain specificity of human antibody responses and inhibitory peptides. We also investigated the domain II loop, a key region involved in inhibitor binding, by comparison of multiple AMA1 crystal structures. Collectively, these results provide valuable insights that should contribute to the design of strain-transcending agents targeting <i>P. falciparum</i> AMA1