646 research outputs found
Cloning, expression, purification and preliminary X-ray diffraction studies of a novel AB5 toxin
No full textAB5 toxins are key virulence factors found in a range of pathogenic bacteria. AB5 toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 Å resolution are reported.Natasha Ng, Dene Littler, Jérôme Le Nours, Adrienne W. Paton, James C. Paton, Jamie Rossjohn and Travis Beddo
Erratum: Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination
No full textIMMUNOLOGY AND INFLAMMATION: Correction for "Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination," by Sophie V. Pageon, Thibault Tabarin, Yui Yamamoto, Yuanqing Ma, John S. Bridgeman, André Cohnen, Carola Benzing, Yijun Gao, Michael D. Crowther, Katie Tungatt, Garry Dolton, Andrew K. Sewell, David A. Price, Oreste Acuto, Robert G. Parton, J. Justin Gooding, Jérémie Rossy, Jamie Rossjohn, and Katharina Gaus, which appeared in issue 37, September 13, 2016, of Proc Natl Acad Sci USA (113:E5454-5463; first published August 29, 2016; 10.1073/pnas.1607436113). The authors note that Philip R. Nicovich should be added to the author list between Yuanqing Ma and John S. Bridgeman. Philip R. Nicovich should be credited with contributing new reagents/analytic tools. The corrected author line, affiliation line, and author contributions appear below. The online version has been corrected
Polymorphism in human cytomegalovirus UL40 impacts on recognition of human leukocyte antigen-E (HLA-E) by natural killer cells
No full textNatural killer (NK) cell recognition of the nonclassical human leukocyte antigen (HLA) molecule HLA-E is dependent on the presentation of a nonamer peptide derived from the leader sequence of other HLA molecules to CD94-NKG2 receptors. However, human cytomegalovirus can manipulate this central innate interaction through the provision of a "mimic" of the HLA-encoded peptide derived from the immunomodulatory glycoprotein UL40. Here, we analyzed UL40 sequences isolated from 32 hematopoietic stem cell transplantation recipients experiencing cytomegalovirus reactivation. The UL40 protein showed a "polymorphic hot spot" within the region that encodes the HLA leader sequence mimic. Although all sequences that were identical to those encoded within HLA-I genes permitted the interaction between HLA-E and CD94-NKG2 receptors, other UL40 polymorphisms reduced the affinity of the interaction between HLA-E and CD94-NKG2 receptors. Furthermore, functional studies using NK cell clones expressing either the inhibitory receptor CD94-NKG2A or the activating receptor CD94-NKG2C identified UL40-encoded peptides that were capable of inhibiting target cell lysis via interaction with CD94-NKG2A, yet had little capacity to activate NK cells through CD94-NKG2C. The data suggest that UL40 polymorphisms may aid evasion of NK cell immunosurveillance by modulating the affinity of the interaction with CD94-NKG2 receptors.Susan L. Heatley, Gabriella Pietra, Jie Lin, Jacqueline M. L. Widjaja, Christopher M. Harpur, Sue Lester, Jamie Rossjohn, Jeff Szer, Anthony Schwarer, Kenneth Bradstock, Peter G. Bardy, Maria Cristina Mingari, Lorenzo Moretta, Lucy C. Sullivan and Andrew G. Brook
Structural basis of subtilase cytotoxin SubAB assembly
No full textPathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.Jérôme Le Nours, Adrienne W. Paton, Emma Byres, Sally Troy, Brock P. Herdman, Matthew D. Johnson, James C. Paton, Jamie Rossjohn, and Travis Beddo
Structure, biological functions and applications of the AB5 toxins
No full textAB5 toxins are important virulence factors for several major bacterial pathogens, including Bordetella pertussis, Vibrio cholerae, Shigella dysenteriae and at least two distinct pathotypes of Escherichia coli. The AB5 toxins are so named because they comprise a catalytic A-subunit, which is responsible for disruption of essential host functions, and a pentameric B-subunit that binds to specific glycan receptors on the target cell surface. The molecular mechanisms by which the AB5 toxins cause disease have been largely unravelled, including recent insights into a novel AB5 toxin family, subtilase cytotoxin (SubAB). Furthermore, AB5 toxins have become a valuable tool for studying fundamental cellular functions, and are now being investigated for potential applications in the clinical treatment of human diseasesTravis Beddoe, Adrienne W. Paton, Jérôme Le Nours, Jamie Rossjohn and James C. Patonhttp://www.elsevier.com/wps/find/journaldescription.cws_home/707424/description#descriptio
Structural insights into the Natural Killer T cell receptor specificity and CD1d-glycolipid recognition
No full textNatural Killer T (NKT) cells play an important role in the immune system as demonstrated by their involvement in tumour surveillance, infection and inflammation. Unlike conventional T cells that recognises peptide antigens when presented by the Major Histocompatibility Complex (pMHC), NKT cells recognise glycolipids, presented by a MHC class I-like molecule (CD1d-α-GalCer) to invoke an immune response. Human semi-invariant NKT cells are unique as the majority of them express a T cell receptor (TCR) bearing an invariant α-chain and restricted β-chain repertoire (Vα24-Jα18;Vβ 11). The mouse orthologue also expresses an invariant α-chain (Vα14-Jα18) but a slightly more diverse β-chain repertoire (Vβ2, Vβ7 and Vβ8.2), where Vβ8.2 is most commonly expressed. The crystal structures of the human NKT TCR-CD1d-α-GalCer and mouse orthologue complexes have provided structural insights into how the NKT TCR can recognise a lipid antigen. While it is clear that the NKT TCR docks CD1d-α-GalCer in a different conformation compared to the TCR-pMHC complexes, there was no clear information on the energetic footprint of the NKT TCR's recognition of CD1d-α-GalCer and also how the NKT TCR can differentiate between closely related α-GalCer analogues to induce a biased cytokine response. In this study, an alanine scanning mutagenesis experiment carried out on the human NKT TCR (Vα24-Jα18;Vβ11) and CD1d, as well as the use of α-GalCer analogues, demonstrated that the Jα18-encoded CDR3α loop and Vβ11-encoded CDR2β loop of the NKT TCR play a crucial role in maintaining its interactions with CD1d-α-GalCer. The minimal usage of only six residues, which are also evolutionary conserved in the mouse NKT TCR (Vα14-Jα18;Vβ8.2), explains the semi-invariant nature of the NKT TCR as well as the basis of NKT cell cross-species reactivity. Furthermore, the interactions these residues made are localised directly above the F′ pocket of CD1d, distal from the galactosyl head group of α-GalCer. The use of α-GalCer analogues that contains glycosyl head group modifications, further demonstrated the lesser energetic contribution the NKT TCR CDR1α loop plays in its interactions with the sugar head group of α-GalCer. In contrast, the NKT TCR CDR3α loop, which makes interactions with α-GalCer as well as CD1d, was determined to be the key CDR loop that is energetically important in glycolipid recognition. NKT cells can differentiate between α-GalCer analogues with small modifications on their glycosyl head group to stimulate a biased T helper (Th) cytokine response. Therefore, the ability of how the mouse Vβ8.2 NKT TCR is able to distinguish between different α-GalCer analogues was further investigated through a combination of biophysical, structural and functional experiments. These data had provided further insight into how NKT cells can recognise and differentiate between structurally similar variants of α-GalCer, also referred to as Altered Glycolipid Ligands (AGLs). The crystal structures of all five NKT TCR-CD1d-AGL complexes revealed minimal structural differences. Variations in terms of affinity and kinetics of the NKT TCR engagement onto CD1d-AGLs as well as differences in cellular responses between AGLs were observed. Modifications on the glycosyl head group of the AGLs, directly impacted NKT cell activation as well as the affinity and t1/2 of the NKT TCR recognition. Furthermore, for these glycosyl head group modified AGLs, ligand potency, as determined by the amount of cytokines produced by the NKT cells, was directly affected by the t1/2 of the NKT TCR-CD1d-AGL interaction. In addition, modifications on the acyl chain of the AGLs do not affect the NKT TCR interaction but reduced NKT cell proliferation. This indicated an alternative antigen processing and presentation pathway for these AGLs by CD1d. On the other hand, truncation of the sphingosine chain resulted in a reduction of NKT TCR affinity resulting in an induced-fit mechanism by the NKT TCR. Collectively, the minimal binding requirements of CD1d restriction as well as the molecular basis of NKT fine specificity in CD1d-AGLs recognition were elucidated
EcxAB is a founding member of a new family of metalloprotease AB(5) toxins with a hybrid cholera-like B subunit
No full textAB5 toxins are composed of an enzymatic A subunit that disrupts cellular function associated with a pentameric B subunit required for host cell invasion. EcxAB is an AB5 toxin isolated from clinical strains of Escherichia coli classified as part of the cholera family due to B subunit homology. Cholera-group toxins have catalytic ADP-ribosyltransferases as their A subunits, so it was surprising that EcxA did not. We confirmed that EcxAB self-associates as a functional toxin and obtained its structure. EcxAB is a prototypical member of a hybrid AB5 toxin family containing metzincin-type metalloproteases as their active A subunit paired to a cholera-like B subunit. Furthermore, EcxA is distinct from previously characterized proteases and thus founds an AB5-associated metzincin family that we term the toxilysins. EcxAB provides the first observation of conserved B subunit usage across different AB5 toxin families and provides evidence that the intersubunit interface of these toxins is far more permissive than previously supposed.Natasha M. Ng, Dene R. Littler, Adrienne W. Paton, Jérôme Le Nours, Jamie Rossjohn, James C. Paton, and Travis Beddo
Structural basis for the association of the HLA-DRB1 locus, citrullination and rheumatoid arthritis
No full textRheumatoid arthritis (RA) is a systemic autoimmune disease affecting approximately 0.5-1% of the Western population. The disease is characterized by synovial joint inflammation, joint pannus and bone erosion, and leads to comorbidities such as lung fibrosis and atherosclerosis, reducing the expected life span of patients by up to 10 years. The human leukocyte antigen (HLA) locus plays a vital role in immunity, encoding highly polymorphic molecules that present peptides to T cell lymphocytes. Whilst the pathogenesis of RA is still unclear, genetic studies have shown that it is associated with the HLA-DRB1 locus. Specifically, the association corresponds to amino acids 70 to 74 in the third hypervariable region of the HLA-DRB1 allele, commonly referred to as the ‘shared epitope’ (SE). These amino acids line the fourth anchoring pocket (P4) of the HLA-DRB1 molecule and therefore have the ability to discriminate which peptides are capable of being presented. In SE allomorphs, the P4 pocket has an overall positive charge, consequently disfavouring positively charged sidechains from occupying this pocket in preference for negatively charged, polar or hydrophobic residues. Interestingly, the SE is associated with the presence of anti-citrullinated protein antibodies (ACPA) in RA patients. Citrullination is the conversion of the positively charged arginine to the polar or neutral citrulline. Previous studies have shown that citrullination of self-antigens can significantly increase the affinity of peptides for SE allomorphs. It is hypothesized that citrullination of peptides in the inflamed joints of RA patients allows these peptides to bind to HLA-DRB1 allomorphs possessing the SE, generating neo-antigens and ultimately causing autoreactivity. However there is no structural data to describe how SE allomorphs preferentially bind to citrullinated self antigens. This thesis presents the structural basis of citrullinated vimentin and aggrecan peptide presentation by the RA susceptible, SE allomorphs, HLA-DRB1*0401 and HLADRB1*0404. Citrulline was accommodated in the positively charged P4 pocket of SE allomorphs, where Lys71β of HLA-DRB1*0401 or Arg71β of HLA-DRB1*0404, performed a key discriminatory role. In addition, Ala74β of the SE motif, provided space for the P4-Cit to adopt an upright configuration, and explains why Ala74β is conserved in all SE allomorphs. In contrast, HLA-DRB1*0402, an RA resistant allomorph that encodes a negatively charged P4 pocket, accommodated both arginine and citrulline. Together, these studies provide a structural basis for the preferential binding of citrullinated peptides to SE allomorphs. The Native American population has an increased prevalence of ACPA+ RA compared to other ethnic groups, and is likely attributable to the high frequency of the SE allele HLADRB1*1402 in this population. HLA-DRB1*1402 exhibits the same SE motif as HLADBR1*0404, and thus may prefer to bind citrulline to arginine in its electropositive P4 pocket. However, HLA-DRB1*1402 also possesses a Ser11β and Ser13β, residues thought to confer resistance to RA. Therefore, we determined the crystal structure of HLADRB1*1402 in complex with native and citrullinated vimentin peptides. Interestingly, the electropositive P4 pocket was able to accommodate both an arginine and a citrulline. However, these residues adopted starkly different conformations, with the arginine buried deep within the P4 pocket, away from the electropositive Arg71β, and instead interacted with Ser11β and Ser13β. In contrast, the P4 citrulline adopted an upright constrained U-type conformation, and formed a hydrogen bond with Arg71β. This result suggests that T cell receptors on T cells could discriminate between the differing configurations of the native and citrullinated peptides. Collectively, these findings provide a molecular basis for the association of HLA Class II SE allomorphs and citrullination in RA pathogenesis. More broadly, this thesis highlights the important role of post-translational modifications in autoimmune disease, and will aid the development of vaccine-based therapies
A structural investigation of human class Ib major histocompatibility complex molecules in innate and adaptive immunity
No full textWhile the Human Leukocyte Antigen (HLA) locus is the most polymorphic region in the human genome, Major Histocompatibility Complex Class Ib (MHC-Ib) molecules display far less polymorphism and variation than the structurally similar MHC Class Ia (MHC-Ia) molecules. From an evolutionary perspective, the polymorphism of MHC-Ia stems from the advantage conferred by a heterozygotic MHC-Ia genotype, due to the recognised role of antigen presentation to clonotypic αβT-cell receptors (TcRs) in the adaptive immune system. In contrast, the primary roles of the various MHC-Ib molecules are in the regulation of the innate immune system, roles which are generally independent of allele variation. Indeed, the two most extensively studied Class Ib molecules in humans, HLA-G and HLA-E, are essentially monomorphic at the amino acid level, though a limited number of polymorphisms have been found in healthy individuals. The role of HLA-G in regulating the innate immune response to the semi-allogeneic foetus in pregnancy has been a recent focus of research in reproductive biology. Little is known about the role of the peptide presented by HLA-G in this context, though the crystal structure of HLA-G presenting the endogenous peptide RIIPRHLQL, has been determined in both monomeric (Clements et al., 2005) and dimeric (Shiroishi et al., 2006a) forms. It has been proposed that the nature of the bound peptide may influence binding to Natural Killer (NK) cell receptors of the Leukocyte Immunoglobulin-like Receptor (LILR/LIR/ILT) family, as well as KIR2DL4, a member of the Killer Immunoglobulin-like Receptor (KIR) family of NK receptors. Therefore, the structure of HLA-G was determined with two further endogenous peptides, KLPAQFYIL and KGPPAALTL, in order to investigate the effect of the bound peptide on the conformation of HLA-G. KIR2DL4, which also displays significantly less polymorphism relative to other members of the KIR family, was expressed recombinantly and studied using biochemical techniques including circular dichroism (CD) analysis and small angle x-ray scattering (SAXS) as part of the wider aim of solving the crystal structure of this NK receptor. HLA-E performs a distinct role in regulating the innate immune response, mediated by the presentation of related peptides, derived from the leader sequence of MHC-I molecules, to NK receptors of the CD94/NKG2 family. This role forms part of the ‘missing self’ reaction, with downregulation of pMHC-I (peptide/MHC-I) production (for example, in infected or damaged cells) resulting in downregulation of pHLA-E presentation and knockout of signals inhibiting NK cell-mediated lysis. Constitutive, activatory signals present in the NK cell are then able to mediate lysis of the infected or damaged cell. While the structure of HLA-E has been solved with several peptides and also in complex with CD94/NKG2A, a role in the adaptive immune system has recently been proposed based on the discovery of HLA-E-restricted T-cell populations in several individuals with latent or resolved Cytomegalovirus (CMV) infections. It has been proposed that the product of the open reading frame UL40 (a Type 1 membrane protein) of some CMV strains has evolved to contain mimotopes of the MHC-I leader sequences, and that in infected individuals whose MHC-I haplotypes do not match the viral mimotopes, a T-cell response may be generated (Ulbrecht et al., 2000). Based on the haplotypes of individuals, a range of non-self pHLA-E-reactive TcRs may be generated. To date, two groups of HLA-E restricted TcR have been characterised, based on peptide recognition patterns (Pietra et al., 2003). The TcRs GF4 (Group 1) and KK50.4 (Group 2) have been isolated as representative of each of these groups. To investigate the mechanism of this TcR:MHC-Ib interaction, the crystal structure of GF4 has been determined in complex with HLA-E presenting the two related peptides VMAPRTLVL and VMAPRTLIL. These structures have enabled comparison with the previously determined KK50.4:HLA-EVMAPRTLIL complex structure (Hoare et al., 2006) as well as those of MHC-Ia:TcR complexes. Collectively, the work presented in this thesis has provided insight into the basis of peptide presentation and subsequent TcR recognition by MHC-Ib molecules in humans
Cover Image
No full textON THE COVERThe antigen-presenting molecule CD1a (white) binds long chain sphingomyelin (blue), which protrudes to the CD1a surface and blocks the approach of T cell receptors (orange and pink). This original image was made by Dr. Erica Tandori, who is an artist in residence at Monash University. She has worked together with Marcin Wegrecki and Jamie Rossjohn to generate an image that captures the main message of the paper. Image © Erica Tandori. https://doi.org/10.1084/jem.2020269
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