RESUMEN
The crystal structure of the Fab fragment from the monoclonal anti-preS2 antibody F124 (IgG1,kappa) has been solved by molecular replacement and refined at 3.0 A resolution. The Fab crystallizes with two independent molecules in the asymmetric unit. F124 recognizes an epitope contained within the preS2 segment between residues 120 and 132 of the surface antigen of hepatitis B virus. The antibody shows a high affinity for the glycan N-linked to Asn123, but it also cross-reacts with the non-glycosylated peptide fragment 120-132. Although crystallization was performed in the presence of an eightfold excess of the cross-reactive peptide, no evidence for the ligand was found in the antigen-binding site, which is close to a neighbouring molecule in the crystal lattice. The antigen-binding site has a groove-like topology which is modulated with pocket-like cavities. It is characterized by a large number of tyrosine and aspartate residues. The importance of germ-line mutations at the binding site is discussed.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos contra la Hepatitis B/química , Antígenos de Superficie de la Hepatitis B/inmunología , Fragmentos Fab de Inmunoglobulinas/química , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Cristalografía por Rayos X , Mutación de Línea Germinal , Anticuerpos contra la Hepatitis B/genética , Fragmentos Fab de Inmunoglobulinas/genética , Ratones , Modelos Moleculares , Conformación Proteica , Electricidad EstáticaRESUMEN
The nucleotide sequence of the monoclonal antibody F124, specific for the preS2 region of the surface antigen of hepatitis B virus, has been determined and an single-chain Fv fragment (scFv) recombinant construction has been cloned and expressed into the periplasmic region of Escherichia coli. The recombinant antibody fragment contains a (Gly4Ser)3 linker connecting the C-terminus of the heavy chain variable region (V(L)) domain to the N-terminus of the light chain variable region (V(L)) domain. A 23-residue peptide segment, containing a c-myc marker for immunochemical detection of the scFv and hexahistidine tag to facilitate its purification, was added C-terminal to the V(L) domain. The scFv mimics the antibody in binding to the native antigen in the form of recombinant hepatitis B surface antigen (HBsAg) particles as well as to peptide fragments carrying the viral epitope.
Asunto(s)
Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Antígenos de Superficie de la Hepatitis B/inmunología , Fragmentos de Inmunoglobulinas/genética , Precursores de Proteínas/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Unión Competitiva , Clonación Molecular , ADN Complementario , Fragmentos de Inmunoglobulinas/inmunología , Datos de Secuencia MolecularRESUMEN
Excess ATP is known to enhance Ca(2+)-ATPase activity and, among other effects, to accelerate the Ca2+ binding reaction. In previous work, we studied the pH dependence of this reaction and proposed a 3H+/2Ca2+ exchange at the transport sites, in agreement with the H+/Ca2+ counter transport. Here we studied the effect of ADP and nonhydrolyzable ATP analogues on the Ca2+ binding reaction at various pH values. At pH 6, where Ca2+ binding is monophasic and slow, ADP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), or adenyl-5'-yl imidodiphosphate (AMPPNP) increased the Ca2+ binding rate constant 20-fold. At pH 7 and 8, where Ca2+ binding is biphasic, the nucleotides induce fast and monophasic Ca2+ binding. At pH 7, AMP-PCP accelerated Ca2+ binding with an apparent dissociation constant of 10 microM. At acidic pH, ADP, AMPPCP, or AMPPNP increased the equilibrium affinity of Ca2+ for ATPase, whereas at alkaline pH, these nucleotides had no effect. At pH 5.5, AMPPCP increased equilibrium Ca2+ binding with an apparent dissociation constant of 1 microM.