Targeting the conserved active site of splicing machines with specific and selective small molecule modulators.

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers.

Revisiting the interaction between complement lectin pathway protease MASP-2 and SARS-CoV-2 nucleoprotein.

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

Interaction Studies of Hexameric and Pentameric IgMs with Serum-Derived C1q and Recombinant C1q Mimetics.

The interaction between IgM and C1q represents the first step of the classical pathway of the complement system in higher vertebrates. To identify the significance of particular IgM/C1q interactions, recombinant IgMs were used in both hexameric and pentameric configurations and with two different specificities, along with C1q derived from human serum (sC1q) and two recombinant single-chain variants of the trimeric globular region of C1q. Interaction and complement activation assays were performed using the ELISA format, and bio-layer interferometry measurements to study kinetic behavior. The differences between hexameric and pentameric IgM conformations were only slightly visible in the interaction assay, but significant in the complement activation assay. Hexameric IgM requires a lower concentration of sC1q to activate the complement compared to pentameric IgM, leading to an increased release of C4 compared to pentameric IgM. The recombinant C1q mimetics competed with sC1q in interaction assays and were able to inhibit complement activation. The bio-layer interferometry measurements revealed KD values in the nanomolar range for the IgM/C1q interaction, while the C1q mimetics exhibited rapid on and off binding rates with the IgMs. Our results make C1q mimetics valuable tools for developing recombinant C1q, specifically its variants, for further scientific studies and clinical applications.

Biophysical Characterization of the Oligomeric States of Recombinant Immunoglobulins Type-M and Their C1q-Binding Kinetics by Biolayer Interferometry.

Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs. But, due to their high oligomeric complexity, in vitro production, biochemical characterization, and biophysical characterization are challenging. In this study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (analytical ultracentrifugation, size exclusion chromatography coupled to multi-angle laser light scattering, mass photometry, and transmission electron microscopy). Each IgM construct is defined by a specific expression and purification pattern with different sample quality. Nevertheless, both purified IgMs were able to activate complement in a C1q-dependent manner. More importantly, BioLayer Interferometry (BLI) was used for characterizing the kinetics of C1q binding to recombinant IgMs. We show that recombinant IgMs possess similar C1q-binding properties as IgMs purified from human plasma.

High-throughput measurements of bone morphogenetic protein/bone morphogenetic protein receptor interactions using biolayer interferometry.

Bone morphogenetic proteins (BMPs) are an important family of growth factors playing a role in a large number of physiological and pathological processes, including bone homeostasis, tissue regeneration, and cancers. In vivo, BMPs bind successively to both BMP receptors (BMPRs) of type I and type II, and a promiscuity has been reported. In this study, we used biolayer interferometry to perform parallel real-time biosensing and to deduce the kinetic parameters (ka, kd) and the equilibrium constant (KD) for a large range of BMP/BMPR combinations in similar experimental conditions. We selected four members of the BMP family (BMP-2, 4, 7, 9) known for their physiological relevance and studied their interactions with five type-I BMP receptors (ALK1, 2, 3, 5, 6) and three type-II BMP receptors (BMPR-II, ACTR-IIA, ACTR-IIB). We reveal that BMP-2 and BMP-4 behave differently, especially regarding their kinetic interactions and affinities with the type-II BMPR. We found that BMP-7 has a higher affinity for the type-II BMPR receptor ACTR-IIA and a tenfold lower affinity with the type-I receptors. While BMP-9 has a high and similar affinity for all type-II receptors, it can interact with ALK5 and ALK2, in addition to ALK1. Interestingly, we also found that all BMPs can interact with ALK5. The interaction between BMPs and both type-I and type-II receptors in a ternary complex did not reveal further cooperativity. Our work provides a synthetic view of the interactions of these BMPs with their receptors and paves the way for future studies on their cell-type and receptor specific signaling pathways.

Molecular Basis of Complement C1q Collagen-Like Region Interaction with the Immunoglobulin-Like Receptor LAIR-1.

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q's CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.

Analysis of the Ligand Recognition Specificities of Human Ficolins Using Surface Plasmon Resonance.

Ficolins are innate immune recognition proteins involved in activation of the lectin complement pathway. These oligomeric lectin-like proteins are assembled from subunits consisting of a collagen-like triple helix and a trimeric fibrinogen-like recognition domain. In humans, three ficolins coexist: they differ in their ligand binding specificities, but share the capacity to associate with proteases through their collagen-like stalks and trigger complement activation. We describe methods to decipher the recognition specificities of ficolins, based on surface plasmon resonance, an optical technique allowing real-time and label-free monitoring of biomolecular interactions. This technique was mainly used to characterize and compare binding of the three recombinant full-length ficolins and of their isolated recognition domains to various immobilized BSA-glycoconjugates, acetylated BSA or biotinylated heparin. The avidity phenomenon that enhances the apparent affinity of interactions between oligomeric lectin-like proteins and the multivalent ligands is also discussed.

Functional recombinant human complement C1q with different affinity tags.

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Hepatitis B virus exploits C-type lectin receptors to hijack cDC1s, cDC2s and pDCs.

OBJECTIVES: C-type lectin receptors (CLRs) are key receptors used by DCs to orchestrate responses to pathogens. During infections, the glycan-lectin interactions shape the virus-host interplay and viruses can subvert the function of CLRs to escape antiviral immunity. Recognition of virus/viral components and uptake by CLRs together with subsequent signalling cascades are crucial in initiating and shaping antiviral immunity, and decisive in the outcome of infection. Yet, the interaction of hepatitis B virus (HBV) with CLRs remains largely unknown. As HBV hijacks DC subsets and viral antigens harbour glycan motifs, we hypothesised that HBV may subvert DCs through CLR binding. METHODS: We investigated here the pattern of CLR expression on BDCA1+ cDC2s, BDCA2+ pDCs and BDCA3+ cDC1s from both blood and liver of HBV-infected patients and explored the ability of HBsAg to bind DC subsets through specific CLRs. RESULTS: We highlighted for the first time that the CLR repertoire of circulating and intrahepatic cDC2s, cDC1s and pDCs was perturbed in patients with chronic HBV infection and that some CLR expression levels correlated with plasma HBsAg and HBV DNA levels. We also identified candidate CLR responsible for HBsAg binding to cDCs (CD367/DCIR/CLEC4A, CD32/FcɣRIIA) and pDCs (CD369/DECTIN1/CLEC7A, CD336/NKp44) and demonstrated that HBsAg inhibited DC functions in a CLR- and glycosylation-dependent manner. CONCLUSION: HBV may exploit CLR pathways to hijack DC subsets and escape from immune control. Such advances bring insights into the mechanisms by which HBV subverts immunity and pave the way for developing innovative therapeutic strategies to restore an efficient immune control of the infection by manipulating the viral glycan-lectin axis.

Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

Structural insights into the substrate recognition and reaction specificity of the PLP-dependent fold-type I isoleucine 2-epimerase from Lactobacillus buchneri.

The isoleucine 2-epimerase from Lactobacillus buchneri has been previously identified and characterized to catalyze the pyridoxal 5'-phosphate (PLP)-dependent racemization and epimerization of a broad spectrum of nonpolar amino acids from L- to D-form and vice versa, in particular isoleucine. In this study, crystal structures of both native and PLP-complex forms of this racemase are presented at 2.6 and 2.15 Å resolution, respectively. Both structures show that the protein belongs to the fold-type I subgroup of PLP-dependent enzymes and is very close to aminobutyrate aminotransferases family, as it has been suspected because of their sequence homology. The extensive structural comparison with fold-type I enzymes with known amino acid racemization activities, including the α-amino-ε-caprolactam racemase from Achromobacter obae and the cystathionine ß-lyase from Escherichia coli, allows us to identify the active site residues responsible for its nonpolar amino acid recognition and reactivity specificity. Our observations also suggest that the racemization reaction by the fold-type I racemases may generally occur thanks to a revised two-base mechanism. Lastly, both structures reveal details on the conformational changes provoked by PLP binding that suggest an induced fit of the active site "entrance door", necessary to accommodate PLP and substrate molecules.

The SH3 regulatory domain of the hematopoietic cell kinase Hck binds ELMO via its polyproline motif.

Eukaryotic EnguLfment and cell MOtility (ELMO) proteins form an evolutionary conserved family of regulators involved in small GTPase dependent actin remodeling processes that regulates the guanine exchange factor activity of some of the Downstream Of CrK (DOCK) family members. Gathered data strongly suggest that DOCK activation by ELMO and the subsequent signaling result from a subtle balance in the binding of partners to ELMO. Among its putative upward modulators, the Hematopoietic cell kinase (Hck), a member of the Src kinase superfamily, has been identified as a binding partner and a specific tyrosine kinase for ELMO1. Indeed, Hck is implicated in distinct molecular signaling pathways governing phagocytosis, cell adhesion, and migration of hematopoietic cells. Although ELMO1 has been shown to interact with the regulatory Src Homology 3 (SH3) domain of Hck, no direct evidence indicating the mode of interaction between Hck and ELMO1 have been provided in the literature. In the present study, we report convergent pieces of evidence that demonstrate the specific interaction between the SH3 domain of Hck and the polyproline motif of ELMO1. Our results also suggest that the tyrosine-phosphorylation state of ELMO1 tail might act as a putative modulator of Hck kinase activity towards ELMO1 that in turn participates in DOCK180 activation and further triggers subsequent signaling towards actin remodeling.

Analysis of relationships between peptide/MHC structural features and naive T cell frequency in humans.

The structural rules governing peptide/MHC (pMHC) recognition by T cells remain unclear. To address this question, we performed a structural characterization of several HLA-A2/peptide complexes and assessed in parallel their antigenicity, by analyzing the frequency of the corresponding Ag-specific naive T cells in A2(+) and A2(-) individuals, as well as within CD4(+) and CD8(+) subsets. We were able to find a correlation between specific naive T cell frequency and peptide solvent accessibility and/or mobility for a subset of moderately prominent peptides. However, one single structural parameter of the pMHC complexes could not be identified to explain each peptide antigenicity. Enhanced pMHC antigenicity was associated with both highly biased TRAV usage, possibly reflecting favored interaction between particular pMHC complexes and germline TRAV loops, and peptide structural features allowing interactions with a broad range of permissive CDR3 loops. In this context of constrained TCR docking mode, an optimal peptide solvent exposed surface leading to an optimal complementarity with TCR interface may constitute one of the key features leading to high frequency of specific T cells. Altogether our results suggest that frequency of specific T cells depends on the fine-tuning of several parameters, the structural determinants governing TCR-pMHC interaction being just one of them.

Potassium acts as a GTPase-activating element on each nucleotide-binding domain of the essential Bacillus subtilis EngA.

EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K(+) binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2.

The C-terminal polyproline-containing region of ELMO contributes to an increase in the life-time of the ELMO-DOCK complex.

The eukaryotic Engulfment and CellMotility (ELMO) proteins form an evolutionary conserved family of key regulators which play a central role in Rho-dependent biological processes such as engulfment and cell motility/migration. ELMO proteins interact with a subset of Downstream of Crk (DOCK) family members, a new type of guanine exchange factors (GEF) for Rac and cdc42 GTPases. The physiological function of DOCK is to facilitate actin remodeling, a process which occurs only in presence of ELMO. Several studies have determined that the last 200 C-terminal residues of ELMO1 and the first 180 N-terminal residues of DOCK180 are responsible for the ELMO-DOCK interaction. However, the precise role of the different domains and motifs identified in these regions has remained elusive. Divergent functional, biochemical and structural data have been reported regarding the contribution of the C-terminal end of ELMO, comprising its polyproline motif, and of the DOCK SH3 domain. In the present study, we have investigated the contribution of the C-terminal end of ELMO1 to the interaction between ELMO1 and the SH3 domain of DOCK180 using nuclear magnetic resonance spectroscopy and surface plasmon resonance. Our data presented here demonstrate the ability of the SH3 domain of DOCK180 to interact with ELMO1, regardless of the presence of the polyproline-containing C-terminal end. However, the presence of the polyproline region leads to a significant increase in the half-life of the ELMO1-DOCK180 complex, along with a moderate increase on the affinity.

Antitumor activity of pyridocarbazole and benzopyridoindole derivatives that inhibit protein kinase CK2.

The alkyloid compound ellipticine derived from the berrywood tree is a topoisomerase II poison that is used in ovarian and breast cancer treatment. In this study, we report the identification of ellipticine derivatives and their tetracyclic angular benzopyridoindole analogues as novel ATP-competitive inhibitors of the protein kinase CK2. In vitro and in vivo assays showed that these compounds have a good pharmacologic profile, causing a marked inhibition of CK2 activity associated with cell cycle arrest and apoptosis in human cancer cells. Further, in vivo assays demonstrate antitumor activity in a mouse xenograft model of human glioblastoma. Finally, crystal structures of CK2-inhibitor complex provide structural insights on the molecular basis of CK2 inhibition. Our work lays the foundation for development of clinically useful CK2 inhibitors derived from a well-studied scaffold with suitable pharmacokinetics parameters.

New potent dual inhibitors of CK2 and Pim kinases: discovery and structural insights.

Protein kinase casein kinase 2 (CK2) is a serine/threonine kinase with evidence of implication in growth dysregulation and apoptosis resistance, making it a relevant target for cancer therapy. Several CK2 inhibitors have been developed showing variable efficiency, emphasizing the need to expand the chemical diversity of those inhibitors. We report the identification and characterization of 2,8-difurandicarboxylic acid derivatives as a new class of nanomolar ATP-competitive inhibitors. Selectivity profiling pointed out proviral insertion Moloney virus kinases (Pim kinases) as the only other kinases that are significantly inhibited. By combining structure-activity relationship analysis with structural determination, we were able to determine the binding mode of these inhibitors for both kinases and to explain their strong inhibitory potency. Essential chemical features necessary for activity on both kinases were then identified. The described compounds are not cell permeable: however, they could provide a lead for developing novel inhibitors usable also in vivo. Given the similar but not redundant pathophysiological functions of CK2 and Pim family members, such inhibitors would provide new attractive leads for targeted cancer therapy. This work highlights that 2 functionally related kinases from different kinome branches display exquisite sensitivity to a common inhibitor.

Crystallization and preliminary X-ray crystallographic characterization of a public CMV-specific TCR in complex with its cognate antigen.

The T-cell response to human cytomegalovirus is characterized by a dramatic reduction of clonal diversity in patients undergoing chronic inflammation or immunodepression. In order to check whether all the selected high-avidity T-cell clones recognize the immunodominant pp65 peptide antigen pp65(495-503) (NLVPMVATV) presented by the major histocompatibility complex (MHC) molecule HLA-A2 in a similar manner, several public high-affinity T-cell receptors (TCRs) specific for the pp65(495-503)-HLA-A2 complex have been investigated. Expression, purification and crystallization were performed and preliminary crystallographic data were collected to 4.7 angstrom resolution for the RA15 TCR in complex with the pp65(495-503)-HLA-A2 complex. Comparison of the RA15-pp65(495-503)-HLA-A2 complex molecular-replacement solution with the structure of another high-affinity pp65(495-503)-HLA-A2-specific TCR, RA14, shows a shared docking mode, indicating that the clonal focusing could be accompanied by the selection of a most favoured peptide-readout mode. However, the position of the RA15 V beta domain is significantly shifted, suggesting a different interatomic interaction network.

Structural bases for the affinity-driven selection of a public TCR against a dominant human cytomegalovirus epitope.

Protective T cell responses elicited along chronic human CMV (HCMV) infections are sometimes dominated by CD8 T cell clones bearing highly related or identical public TCR in unrelated individuals. To understand the principles that guide emergence of these public T cell responses, we have performed structural, biophysical, and functional analyses of an immunodominant public TCR (RA14) directed against a major HLA-A*0201-restricted HCMV Ag (pp65(495-503)) and selected in vivo from a diverse repertoire after chronic stimulations. Unlike the two immunodominant public TCRs crystallized so far, which focused on one peptide hotspot, the HCMV-specific RA14 TCR interacts with the full array of available peptide residues. The conservation of some peptide-MHC complex-contacting amino acids by lower-affinity TCRs suggests a shared TCR-peptide-MHC complex docking mode and supports an Ag-driven selection of optimal TCRs. Therefore, the emergence of a public TCR of an oligoclonal Ag-specific response after repeated viral stimulations is based on a receptor displaying a high structural complementarity with the entire peptide and focusing on three peptide hotspots. This highlights key parameters underlying the selection of a protective T cell response against HCMV infection, which remains a major health issue in patients undergoing bone marrow transplantation.

Steric hindrance and fast dissociation explain the lack of immunogenicity of the minor histocompatibility HA-1Arg Null allele.

The di-allelic HLA-A2 restricted minor histocompatibility Ag HA-1 locus codes for the highly immunogenic HA-1(His) and the nonimmunogenic HA-1(Arg) nonapeptides, differing in one amino acid. The HA-1(His) peptide is currently used for boosting the graft-vs-tumor responses after HLA matched HA-1 mismatched stem cell transplantation; usage of the HA-1(Arg) peptide would significantly enlarge the applicability for this therapy. Our studies on mechanisms causing the HA-1 unidirectional immunogenicity revealed marginal differences in proteasomal digestion, TAP translocation, and binding affinity, whereas both dissociation rates and structural analyses clearly showed marked differences in the stability of these two HLA-A2 bound alleles. These data provide a rationale for the lack of HA-1(Arg) peptide immunogenicity essential for the choice of tumor peptides for stem cell-based immunotherapeutic application.