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1.
J Immunol ; 208(9): 2207-2219, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35428691

RESUMO

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.


Assuntos
Complemento C4b , Anticorpos de Domínio Único , Animais , Ativação do Complemento , Convertases de Complemento C3-C5/metabolismo , Microscopia Crioeletrônica , Humanos , Mamíferos
2.
Proc Natl Acad Sci U S A ; 116(7): 2681-2690, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30679277

RESUMO

Human betacoronaviruses OC43 and HKU1 are endemic respiratory pathogens and, while related, originated from independent zoonotic introductions. OC43 is in fact a host-range variant of the species Betacoronavirus-1, and more closely related to bovine coronavirus (BCoV)-its presumptive ancestor-and porcine hemagglutinating encephalomyelitis virus (PHEV). The ß1-coronaviruses (ß1CoVs) and HKU1 employ glycan-based receptors carrying 9-O-acetylated sialic acid (9-O-Ac-Sia). Receptor binding is mediated by spike protein S, the main determinant of coronavirus host specificity. For BCoV, a crystal structure for the receptor-binding domain S1A is available and for HKU1 a cryoelectron microscopy structure of the complete S ectodomain. However, the location of the receptor-binding site (RBS), arguably the single-most important piece of information, is unknown. Here we solved the 3.0-Å crystal structure of PHEV S1A We then took a comparative structural analysis approach to map the ß1CoV S RBS, using the general design of 9-O-Ac-Sia-binding sites as blueprint, backed-up by automated ligand docking, structure-guided mutagenesis of OC43, BCoV, and PHEV S1A, and infectivity assays with BCoV-S-pseudotyped vesicular stomatitis viruses. The RBS is not exclusive to OC43 and related animal viruses, but is apparently conserved and functional also in HKU1 S1A The binding affinity of the HKU1 S RBS toward short sialoglycans is significantly lower than that of OC43, which we attribute to differences in local architecture and accessibility, and which may be indicative for differences between the two viruses in receptor fine-specificity. Our findings challenge reports that would map the OC43 RBS elsewhere in S1A and that of HKU1 in domain S1B.


Assuntos
Coronavirus Humano OC43/fisiologia , Fusão de Membrana , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Acetilação , Animais , Sítios de Ligação , Humanos , Ratos , Receptores Virais/química
3.
J Struct Biol ; 193(2): 106-14, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26687416

RESUMO

CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf·Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.


Assuntos
Proteínas de Drosophila/química , Fatores de Transcrição/química , Proteínas ral de Ligação ao GTP/química , Cristalografia por Raios X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo
4.
J Biol Chem ; 287(46): 38460-72, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23012363

RESUMO

The NOD-like receptors NAIP5 and NLRC4 play an essential role in the innate immune response to the bacterial tail protein flagellin. Upon flagellin detection, NAIP5 and NLRC4 form a hetero-oligomeric inflammasome that induces caspase-1-dependent cell death. So far, both the mechanism of formation of the NAIP5-NLRC4 inflammasome and its structure are poorly understood. In this study we combine inflammasome reconstitution in HEK293 cells, purification of inflammasome components, and negative stain electron microscopy to address these issues. We find that a Salmonella typhimurium flagellin fragment comprising the D0 domain and the neighboring spoke region is able to co-precipitate NAIP5 and induce formation of the NAIP5-NLRC4 inflammasome. Comparison with smaller fragments indicates that flagellin recognition is mediated by its C-terminal residues as well as the spoke region. We reconstitute the inflammasome from purified flagellin, NAIP5, and NLRC4, thus proving that no other cellular components are required for its formation. Electron micrographs of the purified inflammasome provide unprecedented insight into its architecture, revealing disk-like complexes consisting of 11 or 12 protomers in which NAIP5 and NLRC4 appear to occupy equivalent positions. On the basis of our data, we propose a model for inflammasome formation wherein direct interaction of flagellin with a single NAIP5 induces the recruitment and progressive incorporation of NLRC4, resulting in the formation of a hetero-oligomeric inflammasome.


Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Adaptadoras de Sinalização CARD/química , Proteínas de Ligação ao Cálcio/química , Flagelina/metabolismo , Inflamassomos/metabolismo , Proteína Inibidora de Apoptose Neuronal/química , Salmonella typhimurium/metabolismo , Animais , Caspase 1/química , DNA Complementar/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos , Microscopia Eletrônica/métodos , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína
5.
Biochim Biophys Acta ; 1818(9): 2175-83, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22560898

RESUMO

Golgi-Associated Plant Pathogenesis-Related protein 1 (GAPR-1) is a mammalian protein that belongs to the superfamily of plant pathogenesis related proteins group 1 (PR-1). GAPR-1 is a peripheral membrane-binding protein that strongly associates with lipid-enriched microdomains at the cytosolic leaflet of Golgi membranes. Little is known about the mechanism of GAPR-1 interaction with membranes. We previously suggested that dimerization plays a role in the function of GAPR-1 and here we report that phytic acid (inositol hexakisphosphate) induces dimerization of GAPR-1 in solution. Elucidation of the crystal structure of GAPR-1 in the presence of phytic acid revealed that the GAPR-1 dimer differs from the previously published GAPR-1 dimer structure. In this structure, one of the monomeric subunits of the crystallographic dimer is rotated by 28.5°. To study the GAPR-1 dimerization properties, we investigated the interaction with liposomes in a light scattering assay and by flow cytometry. In the presence of negatively charged lipids, GAPR-1 caused a rapid and stable tethering of liposomes. [D81K]GAPR-1, a mutant predicted to stabilize the IP6-induced dimer conformation, also caused tethering of liposomes. [A68K]GAPR-1 however, a mutant predicted to stabilize the non-rotated dimer conformation, is capable of binding to liposomes but did not cause liposome tethering. Our combined data suggest that the charge properties of the lipid bilayer can regulate GAPR-1 dynamics as a potential mechanism to modulate GAPR-1 function.


Assuntos
Bicamadas Lipídicas/química , Proteínas de Membrana/química , Membrana Celular/metabolismo , Cromatografia em Gel , Cristalografia por Raios X/métodos , Dimerização , Citometria de Fluxo/métodos , Complexo de Golgi/metabolismo , Humanos , Lipídeos/química , Lipossomos/química , Lipossomos/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutação , Fosfatidilinositóis/química , Ácido Fítico/química , Plasmídeos/metabolismo , Conformação Proteica
6.
Nat Struct Mol Biol ; 14(3): 224-8, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17310251

RESUMO

Factor B is the central protease of the complement system of immune defense. Here, we present the crystal structure of human factor B at 2.3-A resolution, which reveals how the five-domain proenzyme is kept securely inactive. The canonical activation helix of the Von Willebrand factor A (VWA) domain is displaced by a helix from the preceding domain linker. The two helices conformationally link the scissile-activation peptide and the metal ion-dependent adhesion site required for binding of the ligand C3b. The data suggest that C3b binding displaces the three N-terminal control domains and reshuffles the two central helices. Reshuffling of the helices releases the scissile bond for final proteolytic activation and generates a new interface between the VWA domain and the serine protease domain. This allosteric mechanism is crucial for tight regulation of the complement-amplification step in the immune response.


Assuntos
Fator B do Complemento/química , Fator B do Complemento/metabolismo , Proteínas do Sistema Complemento/imunologia , Domínio Catalítico , Convertases de Complemento C3-C5/química , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Reguladoras de Ácido Nucleico/genética , Relação Estrutura-Atividade , Fator de von Willebrand/química
7.
Acta Crystallogr Sect E Struct Rep Online ; 67(Pt 3): o586, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21522347

RESUMO

The enanti-opure title compound, C(4)H(10)NO(2) (+)·Cl(-)·H(2)O, forms a two-dimensional network by inter-molecular hydrogen bonding parallel to (010). Non-merohedral twinning with a twofold rotation about the reciprocal c* axis as twin operation was taken into account during intensity integration and structure refinement. This twinning leads to alternative orientations of the stacked hydrogen-bonded layers.

8.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 12): o3026, 2009 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-21578763

RESUMO

In the enanti-opure crystal of the title compound, C(4)H(10)NO(3) (+)·Cl(-), inter-molecular O-H⋯Cl and N-H⋯Cl hydrogen bonds link the mol-ecules into layers parallel to (001).

9.
Structure ; 14(10): 1587-97, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17027507

RESUMO

C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.


Assuntos
Complemento C2a/química , Modelos Moleculares , Aminoácidos/química , Aminoácidos/genética , Domínio Catalítico , Ativação do Complemento , Complemento C2a/genética , Humanos , Ligantes , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato
10.
Nat Commun ; 8(1): 1280, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29097667

RESUMO

Palmitoylation affects membrane partitioning, trafficking and activities of membrane proteins. However, how specificity of palmitoylation and multiple palmitoylations in membrane proteins are determined is not well understood. Here, we profile palmitoylation states of three human claudins, human CD20 and cysteine-engineered prokaryotic KcsA and bacteriorhodopsin by native mass spectrometry. Cysteine scanning of claudin-3, KcsA, and bacteriorhodopsin shows that palmitoylation is independent of a sequence motif. Palmitoylations are observed for cysteines exposed on the protein surface and situated up to 8 Å into the inner leaflet of the membrane. Palmitoylation on multiple sites in claudin-3 and CD20 occurs stochastically, giving rise to a distribution of palmitoylated membrane-protein isoforms. Non-native sites in claudin-3 indicate that membrane-protein function imposed evolutionary restraints on native palmitoylation sites. These results suggest a generic, stochastic membrane-protein palmitoylation process that is determined by the accessibility of palmitoyl-acyl transferases to cysteines on membrane-embedded proteins, and not by a preferred substrate-sequence motif.


Assuntos
Proteínas de Membrana/química , Antígenos CD20/química , Proteínas de Bactérias/química , Bacteriorodopsinas/química , Sítios de Ligação , Claudina-3/química , Claudina-4/química , Claudinas/química , Cisteína/química , Células HEK293 , Humanos , Lipoilação , Espectrometria de Massas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Canais de Potássio/química , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Processos Estocásticos
11.
J Mol Biol ; 326(1): 151-65, 2003 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-12547198

RESUMO

Insect glutathione-S-transferases (GSTs) are grouped in three classes, I, II and recently III; class I (Delta class) enzymes together with class III members are implicated in conferring resistance to insecticides. Class II (Sigma class) GSTs, however, are poorly characterized and their exact biological function remains elusive. Drosophila glutathione S-transferase-2 (GST-2) (DmGSTS1-1) is a class II enzyme previously found associated specifically with the insect indirect flight muscle. It was recently shown that GST-2 exhibits considerable conjugation activity for 4-hydroxynonenal (4-HNE), a lipid peroxidation product, raising the possibility that it has a major anti-oxidant role in the flight muscle. Here, we report the crystal structure of GST-2 at 1.75A resolution. The GST-2 dimer shows the canonical GST fold with glutathione (GSH) ordered in only one of the two binding sites. While the GSH-binding mode is similar to other GST structures, a distinct orientation of helix alpha6 creates a novel electrophilic substrate-binding site (H-site) topography, largely flat and without a prominent hydrophobic-binding pocket, which characterizes the H-sites of other GSTs. The H-site displays directionality in the distribution of charged/polar and hydrophobic residues creating a binding surface that explains the selectivity for amphipolar peroxidation products, with the polar-binding region formed by residues Y208, Y153 and R145 and the hydrophobic-binding region by residues V57, A59, Y211 and the C-terminal V249. A structure-based model of 4-HNE binding is presented. The model suggest that residues Y208, R145 and possibly Y153 may be key residues involved in catalysis.


Assuntos
Drosophila melanogaster/enzimologia , Glutationa Transferase/química , Glutationa Transferase/classificação , Peroxidação de Lipídeos , Aldeídos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Cristalografia por Raios X , Dimerização , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Alinhamento de Sequência
12.
FEBS Lett ; 531(1): 69-73, 2002 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-12401206

RESUMO

Elucidation of the three-dimensional structure of phosphatidylinositol transfer protein alpha (PI-TPalpha) void of phospholipid revealed a site of membrane association connected to a channel for phospholipid binding. Near the top of the channel specific binding sites for the phosphorylcholine and phosphorylinositol head groups were identified. The structure of this open form suggests a mechanism by which PI-TPalpha preferentially binds PI from a membrane interface. Modeling predicts that upon association of PI-TPalpha with the membrane the inositol moiety of bound PI is accessible from the medium. Upon release from the membrane PI-TPalpha adopts a closed structure with the phospholipid bound fully encapsulated. This structure provides new insights as to how PI-TPalpha may play a role in PI metabolism.


Assuntos
Proteínas de Transporte/química , Membrana Celular/enzimologia , Proteínas de Membrana/química , Fosforilcolina/química , Proteínas de Saccharomyces cerevisiae , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Humanos , Camundongos , Modelos Químicos , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos
14.
EMBO J ; 21(9): 2117-21, 2002 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11980708

RESUMO

Phosphatidylinositol transfer protein alpha (PITP alpha) is a ubiquitous and highly conserved protein in multicellular eukaryotes that catalyzes the exchange of phospholipids between membranes in vitro and participates in cellular phospholipid metabolism, signal transduction and vesicular trafficking in vivo. Here we report the three-dimensional crystal structure of a phospholipid-free mouse PITP alpha at 2.0 A resolution. The structure reveals an open conformation characterized by a channel running through the protein. The channel is created by opening the phospholipid-binding cavity on one side by displacement of the C-terminal region and a hydrophobic lipid exchange loop, and on the other side by flattening of the central beta-sheet. The relaxed conformation is stabilized at the proposed membrane association site by hydrophobic interactions with a crystallographically related molecule, creating an intimate dimer. The observed open conformer is consistent with a membrane-bound state of PITP and suggests a mechanism for membrane anchoring and the presentation of phosphatidylinositol to kinases and phospholipases after its extraction from the membrane. Coordinates have been deposited in the Protein Data Bank (accession No. 1KCM).


Assuntos
Proteínas de Transporte/química , Membranas Intracelulares/química , Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Animais , Sítios de Ligação/fisiologia , Proteínas de Transporte/fisiologia , Cristalografia por Raios X , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Camundongos , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos , Estrutura Terciária de Proteína
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