RESUMO
Innate immunity is critical in the early containment of influenza A virus (IAV) infection, and surfactant protein D (SP-D) plays a crucial role in the pulmonary defense against IAV. In pigs, which are important intermediate hosts during the generation of pandemic IAVs, SP-D uses its unique carbohydrate recognition domain (CRD) to interact with IAV. An N-linked CRD glycosylation provides interactions with the sialic acid-binding site of IAV, and a tripeptide loop at the lectin-binding site facilitates enhanced interactions with IAV glycans. Here, to investigate both mechanisms of IAV neutralization in greater detail, we produced an N-glycosylated neck-CRD fragment of porcine SP-D (RpNCRD) in HEK293 cells. X-ray crystallography disclosed that the N-glycan did not alter the CRD backbone structure, including the lectin site conformation, but revealed a potential second nonlectin-binding site for glycans. IAV hemagglutination inhibition, IAV aggregation, and neutralization of IAV infection studies showed that RpNCRD, unlike the human analogue RhNCRD, exhibits potent neutralizing activity against pandemic A/Aichi/68 (H3N2), enabled by both porcine-specific structural features of its CRD. MS analysis revealed an N-glycan site-occupancy of >98% at Asn-303 of RpNCRD with complex-type, heterogeneously branched and predominantly α(2,3)-sialylated oligosaccharides. Glycan-binding array data characterized both RpNCRD and RhNCRD as mannose-type lectins. RpNCRD also bound LewisY structures, whereas RhNCRD bound polylactosamine-containing glycans. The presence of the N-glycan in the CRD increases the glycan-binding specificity of RpNCRD. These insights increase our understanding of porcine-specific innate defense against pandemic IAV and may inform the design of recombinant SP-D-based antiviral drugs.
Assuntos
Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Lectinas/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Ácidos Siálicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Configuração de Carboidratos , Glicosilação , Testes de Inibição da Hemaglutinação , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Polissacarídeos/química , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Homologia de Sequência , SuínosRESUMO
Lung surfactant proteins (SPs) play critical roles in surfactant function and innate immunity. SP-A and SP-D, members of the collectin family of C-type lectins, exhibit distinct ligand specificities, effects on surfactant structure, and host defense functions despite extensive structural homology. SP-A binds to dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SP-D recognize widely divergent pathogen-associated molecular patterns. Previous studies suggested that a ligand-induced surface loop conformational change unique to SP-A contributes to lipid binding affinity. To test this hypothesis and define the structural features of SP-A and SP-D that determine their ligand binding specificities, a structure-guided approach was used to introduce key features of SP-D into SP-A. A quadruple mutant (E171D/P175E/R197N/K203D) that introduced an SP-D-like loop-stabilizing calcium binding site into the carbohydrate recognition domain was found to interconvert SP-A ligand binding preferences to an SP-D phenotype, exchanging DPPC for PI specificity, and resulting in the loss of lipid A binding and the acquisition of more avid mannan binding properties. Mutants with constituent single or triple mutations showed alterations in their lipid and sugar binding properties that were intermediate between those of SP-A and SP-D. Structures of mutant complexes with inositol or methyl-mannose revealed an attenuation of the ligand-induced conformational change relative to wild-type SP-A. These studies suggest that flexibility in a key surface loop supports the distinctive lipid binding functions of SP-A, thus contributing to its multiple functions in surfactant structure and regulation, and host defense.
Assuntos
Modelos Moleculares , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Cinética , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Ligantes , Lipídeo A/química , Lipídeo A/metabolismo , Lipossomos , Mutagênese Sítio-Dirigida , Mutação , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Redobramento de Proteína , Estabilidade Proteica , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.
Assuntos
Cristalografia por Raios X/métodos , Proteolipídeos/metabolismo , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animais , Sítios de Ligação , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Conformação Proteica , Proteína A Associada a Surfactante Pulmonar/genética , RatosRESUMO
We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Proteína D Associada a Surfactante Pulmonar/genética , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Resistência à Doença , Cães , Feminino , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos DBA , Mutação de Sentido Incorreto , Pandemias , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Carga ViralRESUMO
Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. Interactions of SP-D with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with that of full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have determined crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). On the basis of the D325A+R343V-Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding caused by additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V because of alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D-HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Modelos Moleculares , Proteína D Associada a Surfactante Pulmonar/química , Adesividade , Substituição de Aminoácidos , Sítios de Ligação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Vírus da Influenza A Subtipo H3N2/química , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H3N2/metabolismo , Viabilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Conformação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Pigs can act as intermediate hosts by which reassorted influenza A virus (IAV) strains can be transmitted to humans and cause pandemic influenza outbreaks. The innate host defense component surfactant protein D (SP-D) interacts with glycans on the hemagglutinin of IAV and contributes to protection against IAV infection in mammals. This study shows that a recombinant trimeric neck lectin fragment derived from porcine SP-D (pSP-D) exhibits profound inhibitory activity against IAV, in contrast to comparable fragments derived from human SP-D. Crystallographic analysis of the pSP-D fragment complexed with a viral sugar component shows that a unique tripeptide loop alters the lectin site conformation of pSP-D. Molecular dynamics simulations highlight the role of this flexible loop, which adopts a more stable conformation upon sugar binding and may facilitate binding to viral glycans through contact with distal portions of the branched mannoside. The combined data demonstrate that porcine-specific structural features of SP-D contribute significantly to its distinct anti-IAV activity. These findings could help explain why pigs serve as important reservoirs for newly emerging pathogenic IAV strains.
Assuntos
Antivirais/farmacologia , Metabolismo dos Carboidratos , Vírus da Influenza A/efeitos dos fármacos , Proteína D Associada a Surfactante Pulmonar/farmacologia , Animais , Antivirais/química , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cristalização , Primers do DNA , Cães , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , SuínosRESUMO
We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions.
Assuntos
Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/imunologia , Epitopos de Linfócito B/metabolismo , Francisella tularensis/imunologia , Antígenos O/imunologia , Tularemia/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Carga Bacteriana , Sítios de Ligação de Anticorpos , Células Cultivadas , Cristalização , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunidade Humoral , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Conformação Proteica , Tularemia/terapiaRESUMO
Francisella tularensis (Ft), the Gram-negative facultative intracellular bacterium that causes tularemia, is considered a biothreat because of its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS. To further localize the Ab52 epitope and understand the molecular interactions between the antibody and the saccharide, we determined the X-ray crystal structure of the Fab fragment of Ab52 and derived an antibody-antigen complex using molecular docking. The docked complex, refined through energy minimization, reveals an antigen binding site in the shape of a large canyon with a central pocket that accommodates a V-shaped epitope consisting of six sugar residues, α-D-GalpNAcAN(1â4)-α-D-GalpNAcAN(1â3)-ß-D-QuipNAc(1â2)-ß-D-Quip4NFm(1â4)-α-D-GalpNAcAN(1â4)-α-D-GalpNAcAN. These results inform the development of vaccines and immunotherapeutic/immunoprophylactic antibodies against Ft by suggesting a desired topology for binding of the antibody to internal epitopes of Ft LPS. This is the first report of an X-ray crystal structure of a monoclonal antibody that targets a protective Ft B cell epitope.
Assuntos
Anticorpos Monoclonais/química , Francisella tularensis/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Antígenos O/química , Complexo Antígeno-Anticorpo/química , Sequência de Carboidratos , Cristalografia por Raios X , Epitopos , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Antígenos O/imunologia , Conformação ProteicaRESUMO
Surfactant protein A (SP-A), a C-type lectin, plays an important role in innate lung host defense against inhaled pathogens. Crystallographic SP-A·ligand complexes have not been reported to date, limiting available molecular information about SP-A interactions with microbial surface components. This study describes crystal structures of calcium-dependent complexes of the C-terminal neck and carbohydrate recognition domain of SP-A with d-mannose, D-α-methylmannose, and glycerol, which represent subdomains of glycans on pathogen surfaces. Comparison of these complexes with the unliganded SP-A neck and carbohydrate recognition domain revealed an unexpected ligand-associated conformational change in the loop region surrounding the lectin site, one not previously reported for the lectin homologs SP-D and mannan-binding lectin. The net result of the conformational change is that the SP-A lectin site and the surrounding loop region become more compact. The Glu-202 side chain of unliganded SP-A extends out into the solvent and away from the calcium ion; however, in the complexes, the Glu-202 side chain translocates 12.8 Å to bind the calcium. The availability of Glu-202, together with positional changes involving water molecules, creates a more favorable hydrogen bonding environment for carbohydrate ligands. The Lys-203 side chain reorients as well, extending outward into the solvent in the complexes, thereby opening up a small cation-friendly cavity occupied by a sodium ion. Binding of this cation brings the large loop, which forms one wall of the lectin site, and the adjacent small loop closer together. The ability to undergo conformational changes may help SP-A adapt to different ligand classes, including microbial glycolipids and surfactant lipids.
Assuntos
Metabolismo dos Carboidratos , Carboidratos/farmacologia , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/metabolismo , Animais , Cristalografia por Raios X , Glicerol/metabolismo , Glicerol/farmacologia , Lectinas/química , Lectinas/metabolismo , Ligantes , Manose/metabolismo , Manose/farmacologia , Metilmanosídeos/metabolismo , Metilmanosídeos/farmacologia , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína/efeitos dos fármacos , RatosRESUMO
Porcine surfactant protein D (pSP-D) displays distinctively strong, broad-range inhibitory activity against influenza A virus (IAV). N-Linked glycosylation of the carbohydrate recognition domain (CRD) of pSP-D contributes to the high affinity of this collectin for IAV. To investigate the role of the N-linked glycan further, HEK293E protein expression was used to produce recombinant pSP-D (RpSP-D) that has similar structural and antiviral properties as NpSP-D. We introduced an additional N-linked glycan in the CRD of RpSP-D but this modification did not alter the antiviral activity. Human SP-D is unglycosylated in its CRD and less active against IAV compared with pSP-D. In an attempt to modify its antiviral properties, several recombinant human SP-D (RhSP-D) mutants were constructed with N-linked glycans introduced at various locations within its CRD. To retain lectin activity, necessary for the primary interactions between SP-D and IAV, N-linked glycosylation of RhSP-D was shown to be restricted to the corresponding position in the CRD of either pSP-D or surfactant protein A (SP-A). These N-glycosylated RhSP-D mutants, however, did not show increased neutralization activity against IAV. By developing RhSP-D mutants that also have the pSP-D-specific Ser-Gly-Ala loop inserted in the CRD, we could demonstrate that the N-linked glycan-mediated interactions between pSP-D and IAV involves additional structural prerequisites of the pSP-D CRD. Ultimately, these studies will help to develop highly effective SP-D-based therapeutic and prophylactic drugs against IAV.
Assuntos
Vírus da Influenza A/metabolismo , Lectinas , Polissacarídeos/metabolismo , Proteína D Associada a Surfactante Pulmonar/metabolismo , Animais , Cães , Glicosilação , Células HEK293 , Humanos , Vírus da Influenza A/química , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/terapia , Mutação , Polissacarídeos/química , Polissacarídeos/genética , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/genética , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SuínosRESUMO
The recognition of influenza A virus (IAV) by surfactant protein D (SP-D) is mediated by interactions between the SP-D carbohydrate recognition domains (CRD) and glycans displayed on envelope glycoproteins. Although native human SP-D shows potent antiviral and aggregating activity, trimeric recombinant neck+CRDs (NCRDs) show little or no capacity to influence IAV infection. A mutant trimeric NCRD, D325A/R343V, showed marked hemagglutination inhibition and viral neutralization, with viral aggregation and aggregation-dependent viral uptake by neutrophils. D325A/R343V exhibited glucose-sensitive binding to Phil82 hemagglutinin trimer (HA) by surface plasmon resonance. By contrast, there was very low binding to the HA trimer from another virus (PR8) that lacks glycans on the HA head. Mass spectrometry demonstrated the presence of high mannose glycans on the Phil82 HA at positions known to contribute to IAV binding. Molecular modeling predicted an enhanced capacity for bridging interactions between HA glycans and D325A/R343V. Finally, the trimeric D325A/R343V NCRD decreased morbidity and increased viral clearance in a murine model of IAV infection using a reassortant A/WSN/33 virus with a more heavily glycosylated HA. The combined data support a model in which altered binding by a truncated mutant SP-D to IAV HA glycans facilitates viral aggregation, leading to significant viral neutralization in vitro and in vivo. These studies demonstrate the potential utility of homology modeling and protein structure analysis for engineering effective collectin antivirals as in vivo therapeutics.
Assuntos
Resistência à Doença/genética , Evolução Molecular , Vírus da Influenza A Subtipo H1N1/fisiologia , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/genética , Ressonância de Plasmônio de Superfície/métodos , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Cristalografia por Raios X , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/metabolismo , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteína D Associada a Surfactante Pulmonar/metabolismo , Especificidade da EspécieRESUMO
Annexin V (1) specifically binds to phosphatidylserine on apoptotic and necrotic cells as well as certain cancer cells, making it an attractive vehicle for the delivery of therapeutically-relevant conjugates to such sites. The wild-type protein possesses a single thiol at Cys316, which is difficultly accessible to site-specific labeling by simple maleimides. By contrast, 1,4-benzoquinone site-specifically labels annexin V in minutes. The resulting conjugate (5) serves as an intermediate for crosslinking annexin molecules, which can be accomplished within hours either directly for linking annexin V-128 (19), or via an extended sequence involving the crosslinking of two units of (5) by the symmetrical α,ω-dithiol (20). Besides its ability to mediate protein dimer formation while retaining annexin V's ability to bind phosphatidylserine, (5) possesses classic 1,4-benzoquinone reactivity. Various nucleophiles and Diels-Alder dienes form adducts with (5) in reactions that may have general utility for the synthesis of novel biologically active entities. The present work presents the first example of thiol-specific crosslinking of proteins by 1,4-quinone-based methodology designed to exploit the reactivity of this versatile chemical entity.
Assuntos
Anexina A5/química , Benzoquinonas/química , Reagentes de Ligações Cruzadas/química , Multimerização Proteica , Estrutura MolecularRESUMO
Hyperthermophilic archaea have an unusual phosphatase that exhibits activity toward both inositol-1-phosphate and fructose-1,6-bisphosphate, activities carried out by separate gene products in eukaryotes and bacteria. The structures of phosphatases from Archaeoglobus fulgidus (AF2372) and Methanococcus jannaschii (MJ0109), both anaerobic organisms, resemble the dimeric unit of the tetrameric pig kidney fructose bisphosphatase (FBPase). A striking feature of AF2372, but not of MJ0109, is that the sulfhydryl groups of two cysteines, Cys150 and Cys186, are in close proximity (4 A). A similar arrangement of cysteines has been observed in chloroplast FBPases that are regulated by disulfide formation controlled by redox signaling pathways (ferredoxin/thioredoxin). This mode of regulation has not been detected in any other FBPase enzymes. Biochemical assays show that the AF2372 phosphatase activity can be abolished by incubation with O(2). Full activity is restored by incubation with thiol-containing compounds. Neither the C150S variant of AF2372 nor the equivalent phosphatase from M. jannaschii loses activity with oxidation. Oxidation experiments using Escherichia coli thioredoxin, in analogy with the chloroplast FBPase system, indicate an unexpected mode of regulation for AF2372, a key phosphatase in this anaerobic sulfate reducer.
Assuntos
Archaeoglobus fulgidus/enzimologia , Cloroplastos/enzimologia , Frutose-Bifosfatase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Frutose-Bifosfatase/efeitos dos fármacos , Oxidantes/farmacologia , Oxigênio/metabolismo , Monoéster Fosfórico Hidrolases/efeitos dos fármacos , Compostos de Sulfidrila/metabolismo , Tiorredoxinas/farmacologiaRESUMO
The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais Murinos/imunologia , Francisella tularensis/genética , Antígenos O/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/genética , Anticorpos Monoclonais Murinos/genética , Sequência de Bases , Cristalografia por Raios X , Francisella tularensis/imunologia , Imunoensaio , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.
Assuntos
Linfócitos B/imunologia , Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Francisella tularensis/imunologia , Tularemia/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação de Anticorpos , Chaperonina 60/química , Chaperonina 60/genética , Epitopos/genética , Epitopos/imunologia , Camundongos , Dados de Sequência Molecular , Mutação Puntual , Ligação ProteicaRESUMO
Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin-ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D.
Assuntos
Colectinas/metabolismo , Pulmão/metabolismo , Receptores de Reconhecimento de Padrão , Animais , Colectinas/imunologia , Cristalografia por Raios X , Humanos , Imunidade Inata , Pulmão/imunologia , Conformação ProteicaRESUMO
Cleavage of phosphatidylinositol (PI) to inositol 1,2-(cyclic)-phosphate (cIP) and cIP hydrolysis to inositol 1-phosphate by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C are activated by the enzyme binding to phosphatidylcholine (PC) surfaces. Part of this reflects improved binding of the protein to interfaces. However, crystallographic analysis of an interfacially impaired phosphatidylinositol-specific phospholipase (W47A/W242A) suggested protein dimerization might occur on the membrane. In the W47A/W242A dimer, four tyrosine residues from one monomer interact with the same tyrosine cluster of the other, forming a tight dimer interface close to the membrane binding regions. We have constructed mutant proteins in which two or more of these tyrosine residues have been replaced with serine. Phospholipid binding and enzymatic activity of these mutants have been examined to assess the importance of these residues to enzyme function. Replacing two tyrosines had small effects on enzyme activity. However, removal of three or four tyrosine residues weakened PC binding and reduced PI cleavage by the enzyme as well as PC activation of cIP hydrolysis. Crystal structures of Y247S/Y251S in the absence and presence of myo-inositol as well as Y246S/Y247S/Y248S/Y251S indicate that both mutant proteins crystallized as monomers, were very similar to one another, and had no change in the active site region. Kinetic assays, lipid binding, and structural results indicate that either (i) a specific PC binding site, critical for vesicle activities and cIP activation, has been impaired, or (ii) the reduced dimerization potential for Y246S/Y247S/Y248S and Y246S/Y247S/Y248S/Y251S is responsible for their reduced catalytic activity in all assay systems.
Assuntos
Bacillus thuringiensis/enzimologia , Fosfoinositídeo Fosfolipase C/genética , Fosfoinositídeo Fosfolipase C/metabolismo , Substituição de Aminoácidos , Bacillus thuringiensis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Dimerização , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fosfoinositídeo Fosfolipase C/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Tirosina/metabolismoRESUMO
The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.
Assuntos
Bacillus thuringiensis/enzimologia , Fosfoinositídeo Fosfolipase C/química , Fosfoinositídeo Fosfolipase C/metabolismo , Alanina/genética , Alanina/metabolismo , Bacillus thuringiensis/genética , Ativação Enzimática , Modelos Moleculares , Mutação/genética , Fosfoinositídeo Fosfolipase C/genética , Fosfotransferases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7A resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.
Assuntos
Antígenos de Superfície/química , Proteínas do Leite/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X/métodos , Transferência Ressonante de Energia de Fluorescência , Glicina/química , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Fosfolipídeos/química , Homologia de Sequência de Aminoácidos , SoftwareRESUMO
The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp --> Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues.