RESUMEN
Factor H-related protein (FHR) 1 is one of the five human FHRs that share sequence and structural homology with the alternative pathway complement inhibitor FH. Genetic studies on disease associations and functional analyses indicate that FHR-1 enhances complement activation by competitive inhibition of FH binding to some surfaces and immune proteins. We have recently shown that FHR-1 binds to pentraxin 3. In this study, our aim was to investigate whether FHR-1 binds to another pentraxin, C-reactive protein (CRP), analyze the functional relevance of this interaction, and study the role of FHR-1 in complement activation and regulation. FHR-1 did not bind to native, pentameric CRP, but it bound strongly to monomeric CRP via its C-terminal domains. FHR-1 at high concentration competed with FH for CRP binding, indicating possible complement deregulation also on this ligand. FHR-1 did not inhibit regulation of solid-phase C3 convertase by FH and did not inhibit terminal complement complex formation induced by zymosan. On the contrary, by binding C3b, FHR-1 allowed C3 convertase formation and thereby enhanced complement activation. FHR-1/CRP interactions increased complement activation via the classical and alternative pathways on surfaces such as the extracellular matrix and necrotic cells. Altogether, these results identify CRP as a ligand for FHR-1 and suggest that FHR-1 enhances, rather than inhibits, complement activation, which may explain the protective effect of FHR-1 deficiency in age-related macular degeneration.
Asunto(s)
Proteína C-Reactiva/inmunología , Proteína C-Reactiva/metabolismo , Activación de Complemento , Proteínas Inactivadoras del Complemento C3b/inmunología , Proteínas Inactivadoras del Complemento C3b/metabolismo , Sitios de Unión , Proteína C-Reactiva/química , Proteína C-Reactiva/farmacología , Convertasas de Complemento C3-C5 , Complemento C3b/inmunología , Complemento C3b/farmacología , Proteínas Inactivadoras del Complemento C3b/farmacología , Factor H de Complemento , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/inmunología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/inmunología , Humanos , Ligandos , Degeneración Macular/inmunología , Unión Proteica , Componente Amiloide P Sérico/inmunología , Componente Amiloide P Sérico/metabolismoRESUMEN
The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of â¼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C-reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease.
Asunto(s)
Proteína C-Reactiva/metabolismo , Activación de Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Matriz Extracelular/metabolismo , Componente Amiloide P Sérico/metabolismo , Humanos , Ligandos , Unión Proteica , Proteínas RecombinantesRESUMEN
The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.
Asunto(s)
Activación de Complemento/fisiología , Proteínas del Sistema Complemento , Dimerización , Sitios Genéticos , Secuencias de Aminoácidos , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Colapso de la EstructuraRESUMEN
Neisseria meningitidis is a leading cause of sepsis and meningitis. The bacterium recruits factor H (fH), a negative regulator of the complement system, to its surface via fH binding protein (fHbp), providing a mechanism to avoid complement-mediated killing. fHbp is an important antigen that elicits protective immunity against the meningococcus and has been divided into three different variant groups, V1, V2 and V3, or families A and B. However, immunisation with fHbp V1 does not result in cross-protection against V2 and V3 and vice versa. Furthermore, high affinity binding of fH could impair immune responses against fHbp. Here, we investigate a homologue of fHbp in Neisseria gonorrhoeae, designated as Gonococcal homologue of fHbp (Ghfp) which we show is a promising vaccine candidate for N. meningitidis. We demonstrate that Gfhp is not expressed on the surface of the gonococcus and, despite its high level of identity with fHbp, does not bind fH. Substitution of only two amino acids in Ghfp is sufficient to confer fH binding, while the corresponding residues in V3 fHbp are essential for high affinity fH binding. Furthermore, immune responses against Ghfp recognise V1, V2 and V3 fHbps expressed by a range of clinical isolates, and have serum bactericidal activity against N. meningitidis expressing fHbps from all variant groups.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Neisseria gonorrhoeae/inmunología , Neisseria meningitidis Serogrupo A/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Sustitución de Aminoácidos , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Vacunas Meningococicas/genética , Vacunas Meningococicas/inmunología , Neisseria gonorrhoeae/genética , Neisseria meningitidis Serogrupo A/genética , Homología de Secuencia de AminoácidoRESUMEN
The complement system is an essential component of the innate and acquired immune system, and consists of a series of proteolytic cascades that are initiated by the presence of microorganisms. In health, activation of complement is precisely controlled through membrane-bound and soluble plasma-regulatory proteins including complement factor H (fH; ref. 2), a 155 kDa protein composed of 20 domains (termed complement control protein repeats). Many pathogens have evolved the ability to avoid immune-killing by recruiting host complement regulators and several pathogens have adapted to avoid complement-mediated killing by sequestering fH to their surface. Here we present the structure of a complement regulator in complex with its pathogen surface-protein ligand. This reveals how the important human pathogen Neisseria meningitidis subverts immune responses by mimicking the host, using protein instead of charged-carbohydrate chemistry to recruit the host complement regulator, fH. The structure also indicates the molecular basis of the host-specificity of the interaction between fH and the meningococcus, and informs attempts to develop novel therapeutics and vaccines.
Asunto(s)
Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Carbohidratos/química , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Imitación Molecular , Neisseria meningitidis/metabolismo , Sitios de Unión , Factor H de Complemento/inmunología , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Neisseria meningitidis/química , Neisseria meningitidis/inmunología , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB4) within an internal binding pocket. Here, we examined the effect of LTB4 binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB4 in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB4 (C20). We show that the C5 and LTB4 binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB4 binding for full activity. The data highlight the importance of LTB4 in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB4 may be useful for treatment of human immune complex-dependent diseases.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Complejo Antígeno-Anticuerpo/farmacología , Proteínas de Artrópodos/farmacología , Proteínas Portadoras/farmacología , Lipocalinas/farmacología , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/terapia , Animales , Proteínas de Artrópodos/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía de Gases , Complemento C5/metabolismo , Eicosanoides/metabolismo , Ácidos Grasos/metabolismo , Técnicas para Inmunoenzimas , Leucotrieno B4/metabolismo , Lipocalinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/metabolismo , Ovinos , Resonancia por Plasmón de Superficie , Trombina/metabolismoRESUMEN
The complement system is a key component of innate and adaptive immune responses. Complement regulation is critical for prevention and control of disease. We have determined the crystal structure of the complement regulatory enzyme human factor I (fI). FI is in a proteolytically inactive form, demonstrating that it circulates in a zymogen-like state despite being fully processed to the mature sequence. Mapping of functional data from mutants of fI onto the structure suggests that this inactive form is maintained by the noncatalytic heavy-chain allosterically modulating activity of the light chain. Once the ternary complex of fI, a cofactor and a substrate is formed, the allosteric inhibition is released, and fI is oriented for cleavage. In addition to explaining how circulating fI is limited to cleaving only C3b/C4b, our model explains the molecular basis of disease-associated polymorphisms in fI and its cofactors.
Asunto(s)
Factor I de Complemento/química , Factor I de Complemento/genética , Polimorfismo Genético , Estructura Terciaria de Proteína , Regulación Alostérica , Sitios de Unión/genética , Dominio Catalítico , Complemento C3b/química , Complemento C3b/metabolismo , Complemento C4b/química , Complemento C4b/metabolismo , Factor I de Complemento/metabolismo , Cristalización , Cristalografía por Rayos X , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Predisposición Genética a la Enfermedad/genética , Glicosilación , Humanos , Modelos Moleculares , Mutación , Unión ProteicaRESUMEN
The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA5B2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.
Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Flagelos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Flagelos/metabolismo , Flagelos/química , Flagelos/ultraestructura , Cuerpos Basales/metabolismo , Cuerpos Basales/química , Modelos Moleculares , Rotación , Conformación Proteica , Salmonella/metabolismo , Salmonella/química , Salmonella typhimurium/metabolismo , Salmonella typhimurium/químicaRESUMEN
Borrelia burgdorferi is a spirochete responsible for Lyme disease, the most commonly occurring vector-borne disease in Europe and North America. The bacterium utilizes a set of proteins, termed complement regulator-acquiring surface proteins (CRASPs), to aid evasion of the human complement system by recruiting and presenting complement regulator factor H on its surface in a manner that mimics host cells. Presented here is the atomic resolution structure of a member of this protein family, ErpC. The structure provides new insights into the mechanism of recruitment of factor H and other factor H-related proteins by acting as a molecular mimic of host glycosaminoglycans. It also describes the architecture of other CRASP proteins belonging to the OspE/F-related paralogous protein family and suggests that they have evolved to bind specific complement proteins, aiding survival of the bacterium in different hosts.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Borrelia burgdorferi , Proteínas de la Membrana/química , Receptores de Superficie Celular/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/metabolismo , Cristalización , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismoRESUMEN
Borrelia burgdorferi has evolved many mechanisms of evading the different immune systems across its range of reservoir hosts, including the capture and presentation of host complement regulators factor H and factor H-like protein-1 (FHL-1). Acquisition is mediated by a family of complement regulator-acquiring surface proteins (CRASPs), of which the atomic structure of CspA (BbCRASP-1) is known and shows the formation of a homodimeric species which is required for binding. Mutagenesis studies have mapped a putative factor H binding site to a cleft between the two subunits. Presented here is a new atomic structure of CspA which shows a degree of flexibility between the subunits which may be critical for factor H scavenging by increasing access to the binding interface and allows the possibility that the assembly can clamp around the bound complement regulators.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi , Factor H de Complemento/química , Factor H de Complemento/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Cristalografía por Rayos X , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The bacterial flagellum is a macromolecular protein complex that harvests energy from ion-flow across the inner membrane to power bacterial swimming in viscous fluids via rotation of the flagellar filament. Bacteria such as Salmonella enterica are capable of bi-directional flagellar rotation even though ion flow is uni-directional. How uni-directional ion-movement through the inner membrane is utilized by this macromolecular machine to drive bi-directional flagellar rotation is not understood, but a chemotactic response regulator in the cytoplasm is known to reverse the direction of rotation. We here present cryo-EM structures of intact Salmonella flagellar basal bodies, including the cytoplasmic complexes required for power transmission, in conformations representing both directions of rotation. The structures reveal that the conformational changes required for switching the direction of rotation involve 180 degree rotations of both the N- and C-terminal domains of the FliG protein. Combining these models with a new, high-resolution, cryo-EM structure of the MotA5B2 stator, in complex with the C-terminal domain of FliG, reveals how uni-directional ion-flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum.
RESUMEN
Complement receptor 1-related protein Y (CrrY) is an important cell-surface regulator of complement that is unique to rodent species. The structure of rat CrrY domains 1-4 has been determined in two distinct crystal forms and reveals a 70° bend between domains 3 and 4. Comparisons of this structure with those of other complement regulators suggests that rearrangement of this interface may occur on forming the regulatory complex with C3b.
Asunto(s)
Antígenos de Superficie/química , Receptores de Superficie Celular/química , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Ratas , Homología Estructural de ProteínaRESUMEN
The bacterial flagellum is a macromolecular protein complex that enables motility in many species. Bacterial flagella self-assemble a strong, multicomponent drive shaft that couples rotation in the inner membrane to the micrometre-long flagellar filament that powers bacterial swimming in viscous fluids1-3. Here, we present structures of the intact Salmonella flagellar basal body4, encompassing the inner membrane rotor, drive shaft and outer-membrane bushing, solved using cryo-electron microscopy to resolutions of 2.2-3.7 Å. The structures reveal molecular details of how 173 protein molecules of 13 different types assemble into a complex spanning two membranes and a cell wall. The helical drive shaft at one end is intricately interwoven with the rotor component with both the export gate complex and the proximal rod forming interactions with the MS-ring. At the other end, the drive shaft distal rod passes through the LP-ring bushing complex, which functions as a molecular bearing anchored in the outer membrane through interactions with the lipopolysaccharide. The in situ structure of a protein complex capping the drive shaft provides molecular insights into the assembly process of this molecular machine.
Asunto(s)
Cuerpos Basales/ultraestructura , Salmonella/ultraestructura , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cuerpos Basales/metabolismo , Microscopía por Crioelectrón , Flagelos/genética , Flagelos/metabolismo , Flagelos/ultraestructura , Salmonella/genética , Salmonella/metabolismoRESUMEN
Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins (FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.
Asunto(s)
Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Escherichia coli/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Especificidad por Sustrato , Vibrio/metabolismoRESUMEN
Mammalian butyrophilins have various important functions, one for lipid binding but others as ligands for co-inhibition of αß T cells or for stimulation of γδ T cells in the immune system. The chicken BG homologues are dimers, with extracellular immunoglobulin variable (V) domains joined by cysteines in the loop equivalent to complementarity-determining region 1 (CDR1). BG genes are found in three genomic locations: BG0 on chromosome 2, BG1 in the classical MHC (the BF-BL region) and many BG genes in the BG region just outside the MHC. Here, we show that BG0 is virtually monomorphic, suggesting housekeeping function(s) consonant with the ubiquitous tissue distribution. BG1 has allelic polymorphism but minimal sequence diversity, with the few polymorphic residues at the interface of the two V domains, suggesting that BG1 is recognized by receptors in a conserved fashion. Any phenotypic variation should be due to the intracellular region, with differential exon usage between alleles. BG genes in the BG region can generate diversity by exchange of sequence cassettes located in loops equivalent to CDR1 and CDR2, consonant with recognition of many ligands or antigens for immune defence. Unlike the mammalian butyrophilins, there are at least three modes by which BG genes evolve.
Asunto(s)
Proteínas Aviares/genética , Proteínas Aviares/fisiología , Butirofilinas/genética , Butirofilinas/fisiología , ADN Complementario/metabolismo , Variación Genética , Alelos , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Secuencia de Bases , Butirofilinas/química , Pollos , Cromosomas/genética , ADN Complementario/genética , Evolución Molecular , Exones , Modelos Químicos , Glicoproteína Mielina-Oligodendrócito/química , Glicoproteína Mielina-Oligodendrócito/genética , Glicoproteína Mielina-Oligodendrócito/fisiología , ARN/genética , ARN/metabolismoRESUMEN
Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease.
Asunto(s)
Proteínas Bacterianas/metabolismo , Factor H de Complemento/metabolismo , Meningitis Bacterianas/genética , Neisseria meningitidis/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Factor H de Complemento/química , Factor H de Complemento/genética , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Meningitis Bacterianas/inmunología , Datos de Secuencia Molecular , Neisseria meningitidis/patogenicidad , Homología de Secuencia de AminoácidoRESUMEN
The electron paramagnetic resonance technique of double electron-electron resonance (DEER) was used to measure nanometre-scale distances between nitroxide spin labels attached to the complement regulatory protein CD55 (also known as decay accelerating factor) and the von Willebrand factor A (vWF-A) domain of factor B. Following a thorough assessment of the quality of the data, distances obtained from good-quality measurements are compared to predicted distances from a previously hypothesised model for the complex and are found to be incompatible. The success of using these distances as restraints in multi-body docking routines is presented critically.