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1.
J Biol Chem ; 294(45): 17155-17165, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31562242

RESUMO

Bovine conglutinin is an immune protein that is involved in host resistance to microbes and parasites and interacts with complement component iC3b, agglutinates erythrocytes, and neutralizes influenza A virus. Here, we determined the high-resolution (0.97-1.46 Å) crystal structures with and without bound ligand of a recombinant fragment of conglutinin's C-terminal carbohydrate-recognition domain (CRD). The structures disclosed that the high-affinity ligand N-acetyl-d-glucosamine (GlcNAc) binds in the collectin CRD calcium site by interacting with the O3' and O4' hydroxyls alongside additional specific interactions of the N-acetyl group oxygen and nitrogen with Lys-343 and Asp-320, respectively. These residues, unique to conglutinin and differing both in sequence and in location from those in other collectins, result in specific, high-affinity binding for GlcNAc. The binding pocket flanking residue Val-339, unlike the equivalent Arg-343 in the homologous human surfactant protein D, is sufficiently small to allow conglutinin Lys-343 access to the bound ligand, whereas Asp-320 lies in an extended loop proximal to the ligand-binding site and bounded at both ends by conserved residues that coordinate to both calcium and ligand. This loop becomes ordered on ligand binding. The electron density revealed both α and ß anomers of GlcNAc, consistent with the added α/ßGlcNAc mixture. Crystals soaked with α1-2 mannobiose, a putative component of iC3b, reported to bind to conglutinin, failed to reveal bound ligand, suggesting a requirement for presentation of mannobiose as part of an extended physiological ligand. These results reveal a highly specific GlcNAc-binding pocket in conglutinin and a novel collectin mode of carbohydrate recognition.


Assuntos
Acetilglucosamina/metabolismo , Colectinas/química , Colectinas/metabolismo , Soroglobulinas/química , Soroglobulinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica
2.
Infect Immun ; 84(5): 1585-1592, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26953329

RESUMO

The carbohydrate recognition domains (CRDs) of lung collectin surfactant protein D (SP-D) recognize sugar patterns on the surface of lung pathogens and promote phagocytosis. Using Haemophilus influenzae Eagan strains expressing well-characterized lipopolysaccharide (LPS) surface structures of various levels of complexity, we show that bacterial recognition and binding by SP-D is inversely related to LPS chain extent and complexity. The crystal structure of a biologically active recombinant trimeric SP-D CRD complexed with a delipidated Eagan 4A LPS suggests that efficient LPS recognition by SP-D requires multiple binding interactions utilizing the three major ligand-binding determinants in the SP-D binding pocket, with Ca-dependent binding of inner-core heptose accompanied by interaction of anhydro-Kdo (4,7-anhydro-3-deoxy-d-manno-oct-2-ulosonic acid) with Arg343 and Asp325. Combined with enzyme-linked immunosorbent assays (ELISAs) and fluorescence-activated cell sorter (FACS) binding analyses, our results show that extended LPS structures previously thought to be targets for collectins are important in shielding the more vulnerable sites in the LPS core, revealing a mechanism by which pathogens with complex LPS extensions efficiently evade a first-line mucosal innate immune defense. The structure also reveals for the first time the dominant form of anhydro-Kdo.


Assuntos
Haemophilus influenzae/química , Lipopolissacarídeos/química , Proteína D Associada a Surfactante Pulmonar/química , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Lipopolissacarídeos/metabolismo , Ligação Proteica , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
J Biol Chem ; 289(5): 2880-7, 2014 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-24293368

RESUMO

The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn(340) N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.


Assuntos
Quitina/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Quitina/química , Cristalografia por Raios X , Fibrinogênio/química , Humanos , Insetos/citologia , Ligantes , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Relação Estrutura-Atividade
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