Mechanism of chaperone coordination during cotranslational protein folding in bacteria.

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally distinct classes of chaperones promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavored close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor recognizes compact folding intermediates that expose an extensive unfolded surface, and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ, nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to protect incipient structure in the nascent polypeptide well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.

ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity1-3. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown. Several mechanisms, such as those involving lipoprotein particles, exosomes or a specific chaperone, have been proposed to overcome this so-called Wnt solubility problem4-6. Here we provide evidence against these models and show that the Wnt lipid is shielded by the core domain of a subclass of glypicans defined by the Dally-like protein (Dlp). Structural analysis shows that, in the presence of palmitoleoylated peptides, these glypicans change conformation to create a hydrophobic space. Thus, glypicans of the Dlp family protect the lipid of Wnt proteins from the aqueous environment and serve as a reservoir from which Wnt proteins can be handed over to signalling receptors.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

RATIONALE: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. OBJECTIVE: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. METHODS AND RESULTS: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and ß-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/ß-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. CONCLUSIONS: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system.

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3).

CD2AP is an adaptor protein involved in membrane trafficking, with essential roles in maintaining podocyte function within the kidney glomerulus. CD2AP contains three Src homology 3 (SH3) domains that mediate multiple protein-protein interactions. However, a detailed comparison of the molecular binding preferences of each SH3 remained unexplored, as well as the discovery of novel interactors. Thus, we studied the binding properties of each SH3 domain to the known interactor Casitas B-lineage lymphoma protein (c-CBL), conducted a peptide array screen based on the recognition motif PxPxPR and identified 40 known or novel candidate binding proteins, such as RIN3, a RAB5-activating guanine nucleotide exchange factor. CD2AP SH3 domains 1 and 2 generally bound with similar characteristics and specificities, whereas the SH3-3 domain bound more weakly to most peptide ligands tested yet recognized an unusually extended sequence in ALG-2-interacting protein X (ALIX). RIN3 peptide scanning arrays revealed two CD2AP binding sites, recognized by all three SH3 domains, but SH3-3 appeared non-functional in precipitation experiments. RIN3 recruited CD2AP to RAB5a-positive early endosomes via these interaction sites. Permutation arrays and isothermal titration calorimetry data showed that the preferred binding motif is Px(P/A)xPR. Two high-resolution crystal structures (1.65 and 1.11 Å) of CD2AP SH3-1 and SH3-2 solved in complex with RIN3 epitopes 1 and 2, respectively, indicated that another extended motif is relevant in epitope 2. In conclusion, we have discovered novel interaction candidates for CD2AP and characterized subtle yet significant differences in the recognition preferences of its three SH3 domains for c-CBL, ALIX, and RIN3.

Identification of photocrosslinking peptide ligands by mRNA display.

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific, efficient crosslinker can require significant optimisation. We report a modified mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.

Characterisation of a cyclic peptide that binds to the RAS binding domain of phosphoinositide 3-kinase p110α.

P110α is a member of the phosphoinositide 3-kinase (PI3K) enzyme family that functions downstream of RAS. RAS proteins contribute to the activation of p110α by interacting directly with its RAS binding domain (RBD), resulting in the promotion of many cellular functions such as cell growth, proliferation and survival. Previous work from our lab has highlighted the importance of the p110α/RAS interaction in tumour initiation and growth. Here we report the discovery and characterisation of a cyclic peptide inhibitor (cyclo-CRVLIR) that interacts with the p110α-RBD and blocks its interaction with KRAS. cyclo-CRVLIR was discovered by screening a "split-intein cyclisation of peptides and proteins" (SICLOPPS) cyclic peptide library. The primary cyclic peptide hit from the screen initially showed a weak affinity for the p110α-RBD (Kd about 360 µM). However, two rounds of amino acid substitution led to cyclo-CRVLIR, with an improved affinity for p110α-RBD in the low µM (Kd 3 µM). We show that cyclo-CRVLIR binds selectively to the p110α-RBD but not to KRAS or the structurally-related RAF-RBD. Further, using biophysical, biochemical and cellular assays, we show that cyclo-CRVLIR effectively blocks the p110α/KRAS interaction in a dose dependent manner and reduces phospho-AKT levels in several oncogenic KRAS cell lines.

O-Linked Sialoglycans Modulate the Proteolysis of SARS-CoV-2 Spike and Likely Contribute to the Mutational Trajectory in Variants of Concern.

The emergence of a polybasic cleavage motif for the protease furin in SARS-CoV-2 spike has been established as a major factor for human viral transmission. The region N-terminal to that motif is extensively mutated in variants of concern (VOCs). Besides furin, spikes from these variants appear to rely on other proteases for maturation, including TMPRSS2. Glycans near the cleavage site have raised questions about proteolytic processing and the consequences of variant-borne mutations. Here, we identify that sialic acid-containing O-linked glycans on Thr678 of SARS-CoV-2 spike influence furin and TMPRSS2 cleavage and posit O-linked glycosylation as a likely driving force for the emergence of VOC mutations. We provide direct evidence that the glycosyltransferase GalNAc-T1 primes glycosylation at Thr678 in the living cell, an event that is suppressed by mutations in the VOCs Alpha, Delta, and Omicron. We found that the sole incorporation of N-acetylgalactosamine did not impact furin activity in synthetic O-glycopeptides, but the presence of sialic acid reduced the furin rate by up to 65%. Similarly, O-glycosylation with a sialylated trisaccharide had a negative impact on TMPRSS2 cleavage. With a chemistry-centered approach, we substantiate O-glycosylation as a major determinant of spike maturation and propose disruption of O-glycosylation as a substantial driving force for VOC evolution.

An improved method for the incorporation of fluoromethyl ketones into solid phase peptide synthesis techniques.

An improved and expedient technique for the synthesis of peptidyl-fluoromethyl ketones is described. The methodology is based on prior coupling of an aspartate fluoromethyl ketone to a linker and mounting it onto resin-bound methylbenzhydrylamine hydrochloride. Subsequently, by utilising standard Fmoc peptide procedures, a number of short Z-protected peptides were synthesised and assessed as possible inhibitors of the main protease from SARS-CoV-2 (3CLpro).

Fluorescent Amino Acid Initiated de novo Cyclic Peptides for the Label-Free Assessment of Cell Permeability*.

The major obstacle in applying peptides to intracellular targets is their low inherent cell permeability. Standard approaches to attach a fluorophore (e. g. FITC, TAMRA) can change the physicochemical properties of the parent peptide and influence their ability to penetrate and localize in cells. We report a label-free strategy for evaluating the cell permeability of cyclic peptide leads. Fluorescent tryptophan analogues 4-cyanotryptophan (4CNW) and ß-(1-azulenyl)-L-alanine (AzAla) were incorporated into in vitro translated macrocyclic peptides by initiator reprogramming. We then demonstrate these efficient blue fluorescent emitters are good tools for monitoring peptide penetration into cells.

Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-ß responses through SKI stabilization.

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-ß signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-ß signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-ß responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-ß-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity.

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.

The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition.

The Aurora B abscission checkpoint delays cytokinesis until resolution of DNA trapped in the cleavage furrow. This process involves PKCε phosphorylation of Aurora B S227. Assessing if this PKCε-Aurora B module provides a more widely exploited genome-protective control for the cell cycle, we show Aurora B phosphorylation at S227 by PKCε also occurs during mitosis. Expression of Aurora B S227A phenocopies inhibition of PKCε in by-passing the delay and resolution at anaphase entry that is associated with non-disjunction and catenation of sister chromatids. Implementation of this anaphase delay is reflected in PKCε activation following cell cycle dependent cleavage by caspase 7; knock-down of caspase 7 phenocopies PKCε loss, in a manner rescued by ectopically expressing/generating a free PKCε catalytic domain. Molecular dynamics indicates that Aurora B S227 phosphorylation induces conformational changes and this manifests in a profound switch in specificity towards S29 TopoIIα phosphorylation, a response necessary for catenation resolution during mitosis.

Author Correction: The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.

Zoonotic introduction of novel coronaviruses may encounter preexisting immunity in humans. Using diverse assays for antibodies recognizing SARS-CoV-2 proteins, we detected preexisting humoral immunity. SARS-CoV-2 spike glycoprotein (S)-reactive antibodies were detectable using a flow cytometry-based method in SARS-CoV-2-uninfected individuals and were particularly prevalent in children and adolescents. They were predominantly of the immunoglobulin G (IgG) class and targeted the S2 subunit. By contrast, SARS-CoV-2 infection induced higher titers of SARS-CoV-2 S-reactive IgG antibodies targeting both the S1 and S2 subunits, and concomitant IgM and IgA antibodies, lasting throughout the observation period. SARS-CoV-2-uninfected donor sera exhibited specific neutralizing activity against SARS-CoV-2 and SARS-CoV-2 S pseudotypes. Distinguishing preexisting and de novo immunity will be critical for our understanding of susceptibility to and the natural course of SARS-CoV-2 infection.

Molecular determinants regulating selective binding of autophagy adapters and receptors to ATG8 proteins.

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex.

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

Distinct binding modes of two epitopes in Gab2 that interact with the SH3C domain of Grb2.

Grb2 and Gab2 form a complex implicated in normal cell signaling and cancer development. Binding of the Grb2SH3C domain to Gab2 is essential for the interaction, but molecular details remained undefined. Using peptide arrays and isothermal titration calorimetry, two Grb2SH3C binding sites in Gab2 (Gab2a and Gab2b) were confirmed and characterized. Gab2a bears similarity to a p27Kip1 epitope that also binds Grb2SH3C. Crystal structures of both Gab2 epitopes complexed with Grb2SH3C reveal that Gab2b contains a 3(10) helix that positions the arginine and lysine of the core-binding motif RxxK in parallel orientation. In contrast, the Gab2a RxxK motif is embedded in a PPII helix with Arg and Lys in staggered orientation. A similar interaction mode is also present in a new complex of Mona/GadsSH3C with an RxxxxK epitope from the putative phosphatase HD-PTP. In summary, our study reveals interaction types of SH3 domains, highlighting their great versatility.