I02-Macromolecular Crystallography
|
Christos
Stergiou
,
Rhys
Williams
,
Jennifer R.
Fleming
,
Vasiliki
Zouvelou
,
Elpinickie
Ninou
,
Francesca
Andreetta
,
Elena
Rinaldi
,
Ornella
Simoncini
,
Renato
Mantegazza
,
Julius
Bogomolovas
,
John
Tzartos
,
Siegfried
Labeit
,
Olga
Mayans
,
Socrates
Tzartos
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction (NJ) of skeletal muscles. The major MG autoantigen is nicotinic acetylcholine receptor. Other autoantigens at the NJ include MuSK, LRP4 and agrin. Autoantibodies to the intra-sarcomeric striated muscle-specific gigantic protein titin, although not directed to the NJ, are invaluable biomarkers for thymoma and MG disease severity. Thymus and thymoma are critical in MG mechanisms and management. Titin autoantibodies bind to a 30 KDa titin segment, the main immunogenic region (MIR), consisting of an Ig-FnIII-FnIII 3-domain tandem, termed I109-I111. In this work, we further resolved the localization of titin epitope(s) to facilitate the development of more specific anti-titin diagnostics. For this, we expressed protein samples corresponding to 8 MIR and non-MIR titin fragments and tested 77 anti-titin sera for antibody binding using ELISA, competition experiments and Western blots. All anti-MIR antibodies were bound exclusively to the central MIR domain, I110, and to its containing titin segments. Most antibodies were bound also to SDS-denatured I110 on Western blots, suggesting that their epitope(s) are non-conformational. No significant difference was observed between thymoma and non-thymoma patients or between early- and late-onset MG. In addition, atomic 3D-structures of the MIR and its subcomponents were elucidated using X-ray crystallography. These immunological and structural data will allow further studies into the atomic determinants underlying titin-based autoimmunity, improved diagnostics and how to eventually treat titin autoimmunity associated co-morbidities.
|
Feb 2023
|
|
I23-Long wavelength MX
|
Tai-Ying
Chu
,
Céline
Zheng-Gérard
,
Kuan-Yeh
Huang
,
Yu-Chi
Chang
,
Ying-Wen
Chen
,
Kuan-Yu
I
,
Yu-Ling
Lo
,
Nien-Yi
Chiang
,
Hsin-Yi
Chen
,
Martin
Stacey
,
Siamon
Gordon
,
Wen-Yi
Tseng
,
Chiao-Yin
Sun
,
Yen-Mu
Wu
,
Yi-Shin
Pan
,
Chien-Hao
Huang
,
Chun-Yen
Lin
,
Tse-Ching
Chen
,
Kamel
El Omari
,
Marilina
Antonelou
,
Scott R.
Henderson
,
Alan
Salama
,
Elena
Seiradake
,
Hsi-Hsien
Lin
Open Access
Abstract: Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.
|
Oct 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Laura
Del Amo-Maestro
,
Soraia R.
Mendes
,
Arturo
Rodríguez-Banqueri
,
Laura
Garzon-Flores
,
Marina
Girbal
,
María José
Rodríguez-Lagunas
,
Tibisay
Guevara
,
Àngels
Franch
,
Francisco J.
Pérez-Cano
,
Ulrich
Eckhard
,
F. Xavier
Gomis-Rüth
Diamond Proposal Number(s):
[19915]
Open Access
Abstract: The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-β pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded β-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase.
|
Aug 2022
|
|
B21-High Throughput SAXS
Krios IV-Titan Krios IV at Diamond
|
Diamond Proposal Number(s):
[17773, 22724]
Open Access
Abstract: Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.
|
Jul 2022
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Staphylococcus aureus is an opportunistic pathogen that is able to thwart an effective host immune response by producing a range of immune evasion molecules, including S. aureus binder of IgG (Sbi) which interacts directly with the central complement component C3, its fragments and associated regulators. Recently we reported the first structure of a disulfide-linked human C3d17C dimer and highlighted its potential role in modulating B-cell activation. Here we present an X-ray crystal structure of a disulfide-linked human C3d17C dimer, which undergoes a structurally stabilising N-terminal 3D domain swap when in complex with Sbi. These structural studies, in combination with circular dichroism and fluorescence spectroscopic analyses, reveal the mechanism underpinning this unique helix swap event and could explain the origins of a previously discovered N-terminally truncated C3dg dimer isolated from rat serum. Overall, our study unveils a novel staphylococcal complement evasion mechanism which enables the pathogen to harness the ability of dimeric C3d to modulate B-cell activation.
|
May 2022
|
|
I03-Macromolecular Crystallography
|
Changrong
Ge
,
Sylvia
Weisse
,
Bingze
Xu
,
Doreen
Dobritzsch
,
Johan
Viljanen
,
Jan
Kihlberg
,
Nhu-Nguyen
Do
,
Nadine
Schneider
,
Harald
Lanig
,
Rikard
Holmdahl
,
Harald
Burkhardt
Open Access
Abstract: Objectives: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide.Methods: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70289-306, citrullinated CILP982-996 and galactosylated Col2259-273 were determined on cocrystallisation. T cells specific for Col2259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and β-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. Results: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2259-273-specific TCR complexed with DRB1*04:01/Col2259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. Conclusions: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.
|
Mar 2022
|
|
I03-Macromolecular Crystallography
|
Theresa
Kissel
,
Lise
Hafkenscheid
,
Joanneke C.
Kwekkeboom
,
Changrong
Ge
,
Linda M.
Slot
,
Marco
Cavallari
,
Yibo
He
,
Karin A.
Van Schie
,
Rochelle D.
Vergroesen
,
Arieke S. B.
Kampstra
,
Sanne
Reijm
,
Gerrie
Stoeken-Rijsbergen
,
Carolien
Koeleman
,
Lennard M.
Voortman
,
Laura H.
Heitman
,
Bingze
Xu
,
Ger J. M.
Pruijn
,
Manfred
Wuhrer
,
Theo
Rispens
,
Tom W. J.
Huizinga
,
Hans Ulrich
Scherer
,
Michael
Reth
,
Rikard
Holmdahl
,
Rene E. M.
Toes
Diamond Proposal Number(s):
[15806]
Open Access
Abstract: The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.
|
Feb 2022
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[10121]
Open Access
Abstract: Mutations in the adaptor protein PSTPIP1 cause a spectrum of autoinflammatory diseases, including PAPA and PAMI; however, the mechanism underlying these diseases remains unknown. Most of these mutations lie in PSTPIP1 F-BAR domain, which binds to LYP, a protein tyrosine phosphatase associated with arthritis and lupus. To shed light on the mechanism by which these mutations generate autoinflammatory disorders, we solved the structure of the F-BAR domain of PSTPIP1 alone and bound to the C-terminal homology segment of LYP, revealing a novel mechanism of recognition of Pro-rich motifs by proteins in which a single LYP molecule binds to the PSTPIP1 F-BAR dimer. The residues R228, D246, E250, and E257 of PSTPIP1 that are mutated in immunological diseases directly interact with LYP. These findings link the disruption of the PSTPIP1/LYP interaction to these diseases, and support a critical role for LYP phosphatase in their pathogenesis.
|
Feb 2022
|
|
I03-Macromolecular Crystallography
|
Changrong
Ge
,
Dongmei
Tong
,
Erik
Lönnblom
,
Bibo
Liang
,
Weiwei
Cai
,
Cecilia
Fahlquist-Hagert
,
Taotao
Li
,
Alf
Kastbom
,
Inger
Gjertsson
,
Doreen
Dobritzsch
,
Rikard
Holmdahl
Diamond Proposal Number(s):
[8492]
Open Access
Abstract: Objectives: Cartilage oligomeric matrix protein (COMP) is an autoantigen in rheumatoid arthritis and experimental arthritis models. We aimed to investigate the structure, function and relevance of anti-COMP antibodies. Methods: The pathogenicity of monoclonal anti-COMP antibodies in mice was investigated by passive transfer experiments and interaction with cartilage by immunohistochemical staining. The interaction of the monoclonal antibody 15A11 in complex with its specific COMP epitope P6 was determined by X-ray crystallography. The modulation of the binding to 15A11 by calcium ions was studied by ELISA and the surface plasma resonance technique. The clinical relevance and value of serum IgG specific for the COMP P6 epitope and its citrullinated variants were evaluated in a large Swedish cohort of RA patients. Results: The murine monoclonal anti-COMP antibody 15A11 induced arthritis in naïve mice. The crystal structure of 15A11-P6 complex explained how the antibody could bind to COMP, which can be modulated by calcium ions. Moreover, serum IgGs specific for the COMP P6 peptide and its citrullinated variants were detectable at significantly higher levels in RA patients compared to the healthy controls and correlated with a higher disease activity score. Conclusions: We provide the structural basis for binding a pathogenic anti-COMP antibody to cartilage. The recognized epitope can be citrullinated and levels of antibodies to this epitope are elevated in RA patients and correlate with higher disease activity, implicating a pathogenic role of anti-COMP antibodies in a subset of RA patients.
|
Jan 2022
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[17434]
Open Access
Abstract: Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.
|
Nov 2021
|
|
