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1.
Cell ; 180(5): 878-894.e19, 2020 03 05.
Article in English | MEDLINE | ID: mdl-32059783

ABSTRACT

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.


Subject(s)
Autoantibodies/genetics , Autoimmune Diseases/genetics , B-Lymphocytes/immunology , Lymphoma/genetics , Animals , Autoantibodies/immunology , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , B-Lymphocytes/pathology , CARD Signaling Adaptor Proteins/genetics , Carrier Proteins/genetics , Clonal Evolution/genetics , Clonal Evolution/immunology , Cyclin D3/genetics , Guanylate Cyclase/genetics , Humans , Immediate-Early Proteins/genetics , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/immunology , Inhibitor of Differentiation Proteins/genetics , Lymphoma/immunology , Lymphoma/pathology , Mice , Mutation/genetics , Mutation/immunology , Neoplasm Proteins/genetics , Sequence Analysis, DNA/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Tumor Suppressor Proteins/genetics , V(D)J Recombination/genetics
2.
Nat Immunol ; 20(10): 1299-1310, 2019 10.
Article in English | MEDLINE | ID: mdl-31534238

ABSTRACT

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.


Subject(s)
Poxviridae Infections/immunology , Poxviridae/physiology , Protein Domains/genetics , Tumor Necrosis Factor alpha-Induced Protein 3/genetics , Alleles , Animals , Extinction, Biological , Humans , Immunity , Inflammation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation, Missense/genetics , Phosphorylation
3.
Immunity ; 54(12): 2908-2921.e6, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34788600

ABSTRACT

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.


Subject(s)
Betacoronavirus/physiology , COVID-19 Vaccines/immunology , Coronavirus Infections/immunology , Severe acute respiratory syndrome-related coronavirus/physiology , Spike Glycoprotein, Coronavirus/metabolism , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Conserved Sequence/genetics , Evolution, Molecular , Humans , Immunization , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccine Development
4.
Proc Natl Acad Sci U S A ; 119(28): e2123212119, 2022 07 12.
Article in English | MEDLINE | ID: mdl-35867757

ABSTRACT

Humans lack the capacity to produce the Galα1-3Galß1-4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.


Subject(s)
Anaphylaxis , Antibodies , Food Hypersensitivity , Immunoglobulin Heavy Chains , Immunoglobulin Variable Region , Tick-Borne Diseases , Trisaccharides , Anaphylaxis/immunology , Animals , Antibodies/chemistry , Antibodies/genetics , Antibody Formation/genetics , Antigen-Antibody Complex/chemistry , Crystallography, X-Ray , Food Hypersensitivity/immunology , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Mice , Mice, Knockout , Peptide Library , Protein Conformation , Tick-Borne Diseases/immunology , Trisaccharides/genetics , Trisaccharides/immunology
5.
Proc Natl Acad Sci U S A ; 117(36): 22341-22350, 2020 09 08.
Article in English | MEDLINE | ID: mdl-32855302

ABSTRACT

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.


Subject(s)
Antibodies , Antibody Diversity/genetics , Autoantigens , Gene Rearrangement, B-Lymphocyte/genetics , Mutation/genetics , Animals , Antibodies/chemistry , Antibodies/genetics , Antibodies/metabolism , Antibody Affinity/genetics , Autoantibodies/chemistry , Autoantibodies/genetics , Autoantibodies/metabolism , Autoantigens/chemistry , Autoantigens/metabolism , Complementarity Determining Regions/genetics , Immunity, Humoral/genetics , Mice , Models, Molecular , Protein Conformation , Somatic Hypermutation, Immunoglobulin/genetics
6.
J Clin Immunol ; 41(6): 1272-1290, 2021 08.
Article in English | MEDLINE | ID: mdl-33929673

ABSTRACT

Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5-7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.


Subject(s)
Interleukin-21 Receptor alpha Subunit/deficiency , Interleukin-21 Receptor alpha Subunit/genetics , Adolescent , B-Lymphocytes/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Child , Child, Preschool , Cryptosporidiosis/genetics , Cryptosporidiosis/immunology , Cryptosporidium/immunology , Female , Genomics/methods , Humans , Immunity, Humoral/genetics , Immunity, Humoral/immunology , Infant , Interleukin-21 Receptor alpha Subunit/immunology , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Male , Memory B Cells/immunology , Persistent Infection/genetics , Persistent Infection/immunology , Phenotype , Signal Transduction/genetics , Signal Transduction/immunology , Young Adult
7.
Proc Natl Acad Sci U S A ; 114(15): 3897-3902, 2017 04 11.
Article in English | MEDLINE | ID: mdl-28356519

ABSTRACT

Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.


Subject(s)
Evolution, Molecular , Phylogeny , Receptors, Polymeric Immunoglobulin/chemistry , Animals , Crystallography, X-Ray , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin kappa-Chains/chemistry , Immunoglobulin kappa-Chains/genetics , Muramidase/chemistry , Receptors, Polymeric Immunoglobulin/genetics , Vertebrates/genetics , Vertebrates/immunology
8.
Biochemistry ; 58(13): 1701-1704, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30900875

ABSTRACT

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.


Subject(s)
Antibodies, Bispecific/immunology , Peptide Library , Amino Acid Sequence , Antibodies, Bispecific/chemistry , Antibody Affinity , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , Models, Molecular
9.
BMC Struct Biol ; 18(1): 10, 2018 08 22.
Article in English | MEDLINE | ID: mdl-30134879

ABSTRACT

BACKGROUND: Lysozyme purified from duck eggs (DEL) has long been used as a model antigen as a counterpoint to the enzyme purified from hen eggs (HEL). However, unlike the single C-type variant found in hen eggs, duck eggs contain multiple isoforms: I, II and III. We recently reported the structures of isoforms I and III from Pekin duck (Anas platyrhynchos) and unequivocally determined the sequences of all three isoforms by mass spectrometry. Here we present the crystal structure of isoform II (DEL-II). RESULTS: Lysozyme isoform II was purified from isoforms I and III using ion-exchange and gel-filtration chromatography, then crystallized. X-ray diffraction data were collected to 1.15 Å resolution and the structure of DEL-II was solved by molecular replacement using the structure of DEL-I as the search model. It contains two molecules in the crystallographic asymmetric unit: both molecules display a canonical C-type lysozyme fold and electron density consistent with the expected sequence. The most significant difference between the two molecules concerns different conformations of a surface loop containing one of the expected amino acid differences between the isoforms. CONCLUSIONS: The structure of DEL-II supports the primary sequence as elucidated by a combination of amino acid sequencing, DNA sequencing and mass spectrometry, with strong electron density confirming it to be an S37G G71R variant of DEL I, and differing from hen egg lysozyme at a total of 21 amino acid positions.


Subject(s)
Ducks/metabolism , Egg Proteins/chemistry , Muramidase/chemistry , Animals , Avian Proteins/chemistry , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Protein Isoforms/chemistry , X-Ray Diffraction
10.
Nat Commun ; 14(1): 687, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36755042

ABSTRACT

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Female , Animals , Mice , Broadly Neutralizing Antibodies , Leukocytes, Mononuclear , Antibodies, Viral , Antibodies, Monoclonal , Antibodies, Neutralizing , Epitopes , Spike Glycoprotein, Coronavirus/genetics , Neutralization Tests
11.
J Biol Chem ; 286(50): 42971-80, 2011 Dec 16.
Article in English | MEDLINE | ID: mdl-22025611

ABSTRACT

Combinations of LIM homeodomain proteins form a transcriptional "LIM code" to direct the specification of neural cell types. Two paralogous pairs of LIM homeodomain proteins, LIM homeobox protein 3/4 (Lhx3/Lhx4) and Islet-1/2 (Isl1/Isl2), are expressed in developing ventral motor neurons. Lhx3 and Isl1 interact within a well characterized transcriptional complex that triggers motor neuron development, but it was not known whether Lhx4 and Isl2 could participate in equivalent complexes. We have identified an Lhx3-binding domain (LBD) in Isl2 based on sequence homology with the Isl1(LBD) and show that both Isl2(LBD) and Isl1(LBD) can bind each of Lhx3 and Lhx4. X-ray crystal- and small-angle x-ray scattering-derived solution structures of an Lhx4·Isl2 complex exhibit many similarities with that of Lhx3·Isl1; however, structural differences supported by mutagenic studies reveal differences in the mechanisms of binding. Differences in binding have implications for the mode of exchange of protein partners in transcriptional complexes and indicate a divergence in functions of Lhx3/4 and Isl1/2. The formation of weaker Lhx·Isl complexes would likely be masked by the availability of the other Lhx·Isl complexes in postmitotic motor neurons.


Subject(s)
Transcription Factors/metabolism , Animals , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , LIM Domain Proteins/chemistry , LIM Domain Proteins/genetics , LIM Domain Proteins/metabolism , LIM-Homeodomain Proteins/chemistry , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Mice , Mutagenesis , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/genetics , Two-Hybrid System Techniques
12.
J Biol Chem ; 286(44): 38439-38447, 2011 Nov 04.
Article in English | MEDLINE | ID: mdl-21917915

ABSTRACT

Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.


Subject(s)
Antigens, Bacterial/chemistry , Hemoglobins/chemistry , Receptors, Cell Surface/chemistry , Staphylococcal Infections/microbiology , Staphylococcus aureus/metabolism , Antigens, Bacterial/metabolism , Bacterial Proteins/chemistry , Crystallography, X-Ray/methods , Humans , Iron/chemistry , Ligands , Light , Molecular Conformation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cell Surface/metabolism , Spectrophotometry, Ultraviolet/methods , Staphylococcal Infections/metabolism
13.
Neuropeptides ; 92: 102231, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35180645

ABSTRACT

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) form the evolutionarily conserved pancreatic polypeptide family. While the fold is widely utilized in nature, crystal structures remain elusive, particularly for the human forms, with only the structure of a distant avian form of PP reported. Here we utilize a crystallization chaperone (antibody Fab fragment), specifically recognizing the amidated peptide termini, to solve the structures of human NPY and human PYY. Intriguingly, and despite limited sequence identity (~50%), the structure of human PYY closely resembles that of avian PP, highlighting the broad structural conservation of the fold throughout evolution. Specifically, the PYY structure is characterized by a C-terminal amidated α-helix, preceded by a backfolded poly-proline N-terminus, with the termini in close proximity to each other. In contrast, in the structure of human NPY the N-terminal component is disordered, while the helical component of the peptide is observed in a four-helix bundle type arrangement, consistent with a propensity for multimerization suggested by NMR studies.


Subject(s)
Neuropeptide Y , Peptide YY , Humans , Pancreatic Polypeptide , Receptors, Neuropeptide Y
14.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 2): 105-11, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21245531

ABSTRACT

The Thermus thermophilus protein TTHA0988 is a protein of unknown function which represents a fusion of two proteins found almost ubiquitously across the bacterial kingdom. These two proteins perform a role regulating sporulation in Bacillus subtilis, where they are known as KipI and KipA. kipI and kipA genes are usually found immediately adjacent to each other and are often fused to produce a single polypeptide, as is the case with TTHA0988. Here, three crystal forms are reported of TTHA0988, the first structure to be solved from the family of `KipI-KipA fusion' proteins. Comparison of the three forms reveals structural flexibility which can be described as a hinge motion between the `KipI' and `KipA' components. TTHA0988 is annotated in various databases as a putative allophanate hydrolase. However, no such activity could be identified and genetic analysis across species with known allophanate hydrolases indicates that a misannotation has occurred.


Subject(s)
Bacterial Proteins/chemistry , Thermus thermophilus/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , Models, Molecular , Protein Binding , Protein Structure, Quaternary , Structural Homology, Protein , Thermus thermophilus/metabolism
15.
Elife ; 102021 10 27.
Article in English | MEDLINE | ID: mdl-34704555

ABSTRACT

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.


Subject(s)
Antigen Presentation/physiology , Antigens, Surface , B-Lymphocytes/immunology , Receptors, Antigen, B-Cell/immunology , T-Lymphocytes/immunology , Animals , Cell Line , Cell Membrane , Exocytosis , Lysosomes/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Permeability
16.
MAbs ; 13(1): 1922134, 2021.
Article in English | MEDLINE | ID: mdl-34024246

ABSTRACT

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Severe acute respiratory syndrome-related coronavirus/immunology , Antibody Specificity , Cross Reactions , Humans , Mutagenesis, Site-Directed
17.
Acta Crystallogr D Biol Crystallogr ; 65(Pt 6): 574-81, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19465772

ABSTRACT

The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.


Subject(s)
Bacillus subtilis/enzymology , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Protein Kinases/chemistry , Selenomethionine/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cloning, Molecular , Crystallization , DNA-Binding Proteins/metabolism , Histidine Kinase , Magnetic Resonance Imaging , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Phosphotransferases/chemistry , Phosphotransferases/metabolism , Protein Binding , Protein Conformation , Protein Kinases/metabolism , Protein Multimerization , Scattering, Small Angle , Selenium Radioisotopes/metabolism
18.
Article in English | MEDLINE | ID: mdl-19194008

ABSTRACT

A stable intramolecular complex comprising the LIM domains of the LIM-homeodomain protein Lhx4 tethered to a peptide region of Isl2 has been engineered, purified and crystallized. The monoclinic crystals belonged to space group P2(1), with unit-cell parameters a = 46.8, b = 88.7, c = 49.9 A, beta = 111.9 degrees, and diffracted to 2.16 A resolution.


Subject(s)
Homeodomain Proteins/chemistry , Homeodomain Proteins/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , X-Ray Diffraction , Animals , Crystallization , LIM-Homeodomain Proteins , Mice , Motor Neurons/chemistry , Motor Neurons/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Binding/physiology , Protein Engineering/methods
19.
Protein Eng Des Sel ; 32(8): 359-366, 2019 12 31.
Article in English | MEDLINE | ID: mdl-31641749

ABSTRACT

Staphylococcus aureus interacts with the human immune system through the production of secreted factors. Key among these is protein A, a B-cell superantigen capable of interacting with both antibody Fc and VH regions. Here, we review structural and molecular features of this important example of naturally occurring bacterial superantigens, as well as engineered variants and their application in biotechnology.


Subject(s)
Immunoglobulin Fc Fragments/immunology , Staphylococcal Protein A/immunology , Staphylococcus aureus/immunology , Superantigens/immunology , Amino Acid Sequence , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Protein Binding , Protein Engineering/methods , Protein Folding , Sequence Homology, Amino Acid , Staphylococcal Protein A/chemistry , Staphylococcal Protein A/genetics , Staphylococcus aureus/genetics , Staphylococcus aureus/metabolism , Superantigens/chemistry , Superantigens/genetics
20.
J Am Chem Soc ; 130(25): 8069-78, 2008 Jun 25.
Article in English | MEDLINE | ID: mdl-18507382

ABSTRACT

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active ("off-Cu") conformation, and the side chain of the so-called "gate" residue Tyr296 is consistently in the "gate-open" conformation. Among the wires tested, the most stable complex is produced when the wire has a -(CH2)4- linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.


Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Arthrobacter/enzymology , Ruthenium/chemistry , Binding Sites , Crystallography, X-Ray , Kinetics , Models, Molecular , Protein Binding , Stereoisomerism , Substrate Specificity
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