I04-Macromolecular Crystallography
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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.
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May 2022
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I03-Macromolecular Crystallography
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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.
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Mar 2022
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I03-Macromolecular Crystallography
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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.
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Feb 2022
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I03-Macromolecular Crystallography
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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.
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Feb 2022
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I03-Macromolecular Crystallography
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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.
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Jan 2022
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Krios I-Titan Krios I at Diamond
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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.
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Nov 2021
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana S.
Luis
,
Chunsheng
Jin
,
Gabriel
Vasconcelos Pereira
,
Robert W. P.
Glowacki
,
Sadie R.
Gugel
,
Shaleni
Singh
,
Dominic P.
Byrne
,
Nicholas A.
Pudlo
,
James A.
London
,
Arnaud
Basle
,
Mark
Reihill
,
Stefan
Oscarson
,
Patrick A.
Eyers
,
Mirjam
Czjzek
,
Gurvan
Michel
,
Tristan
Barbeyron
,
Edwin A.
Yates
,
Gunnar C.
Hansson
,
Niclas G.
Karlsson
,
Alan
Cartmell
,
Eric C.
Martens
Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease.
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Oct 2021
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I04-Macromolecular Crystallography
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Femke A.
Meijer
,
Annet O. W. M.
Saris
,
Richard G.
Doveston
,
Guido J. M.
Oerlemans
,
Rens M. J. M.
De Vries
,
Bente A.
Somsen
,
Anke
Unger
,
Bert
Klebl
,
Christian
Ottmann
,
Peter J.
Cossar
,
Luc
Brunsveld
Diamond Proposal Number(s):
[27960]
Open Access
Abstract: The inhibition of the nuclear receptor retinoic-acid-receptor-related orphan receptor γt (RORγt) is a promising strategy in the treatment of autoimmune diseases. RORγt features an allosteric binding site within its ligand-binding domain that provides an opportunity to overcome drawbacks associated with orthosteric modulators. Recently, trisubstituted isoxazoles were identified as a novel class of allosteric RORγt inverse agonists. This chemotype offers new opportunities for optimization into selective and efficacious allosteric drug-like molecules. Here, we explore the structure–activity relationship profile of the isoxazole series utilizing a combination of structure-based design, X-ray crystallography, and biochemical assays. The initial lead isoxazole (FM26) was optimized, resulting in compounds with a ∼10-fold increase in potency (low nM), significant cellular activity, promising pharmacokinetic properties, and a good selectivity profile over the peroxisome-proliferated-activated receptor γ and the farnesoid X receptor. We envisage that this work will serve as a platform for the accelerated development of isoxazoles and other novel chemotypes for the effective allosteric targeting of RORγt.
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May 2021
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I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: The folding of disulfide bond containing proteins in the endoplasmic reticulum (ER) is a complex process that requires protein folding factors, some of which are protein-specific. The ER resident saposin-like protein pERp1 (MZB1, CNPY5) is crucial for the correct folding of IgA, IgM and integrins. pERp1 also plays a role in ER calcium homeostasis and plasma cell mobility. As an important factor for proper IgM maturation and hence immune function, pERp1 is upregulated in many auto-immune diseases. This makes it a potential therapeutic target. pERp1 belongs to the CNPY family of ER resident saposin-like proteins. To date, five of these proteins have been identified. All are implicated in protein folding and all contain a saposin-like domain. All previously structurally characterized saposins are involved in lipid binding. However, there are no reports of CNPY family members interacting with lipids, suggesting a novel function for the saposin fold. However, the molecular mechanisms of their function remain elusive. To date, no structure of any CNPY protein has been reported. Here, we present the high-resolution (1.4 Å) crystal structure of human pERp1 and confirm that it has a saposin-fold with unique structural elements not present in other saposin-fold structures. The implications for the role of CNPY proteins in protein folding in the ER are discussed.
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Jan 2021
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I03-Macromolecular Crystallography
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Sandra
Röhm
,
Martin
Schroeder
,
Jessica E.
Dwyer
,
Caroline S.
Widdowson
,
Apirat
Chaikuad
,
Benedict-Tilman
Berger
,
Andreas C.
Joerger
,
Andreas
Krämer
,
Jule
Harbig
,
Daniel
Dauch
,
Mark
Kudolo
,
Stefan
Laufer
,
Mark C.
Bagley
,
Stefan
Knapp
Diamond Proposal Number(s):
[10619]
Abstract: The p38 MAPK cascade is a key signaling pathway linked to a multitude of physiological functions and of central importance in inflammatory and autoimmune diseases. Although studied extensively, little is known about how conformation-specific inhibitors alter signaling outcomes. Here, we have explored the highly dynamic back pocket of p38 MAPK with allosteric urea fragments. However, screening against known off-targets showed that these fragments maintained the selectivity issues of their parent compound BIRB-796, while combination with the hinge-binding motif of VPC-00628 greatly enhanced inhibitor selectivity. Further efforts focused therefore on the exploration of the αC-out pocket of p38 MAPK, yielding compound 137 as a highly selective type-II inhibitor. Even though 137 is structurally related to a recent p38 type-II chemical probe, SR-318, the data presented here provide valuable insights into back-pocket interactions that are not addressed in SR-318 and it provides an alternative chemical tool with good cellular activity targeting also the p38 back pocket.
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Dec 2020
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