Crystallization and preliminary X-ray analysis of PepC, a thiol aminopeptidase from Lactoccocus lactis homologous to bleomycin hydrolase.

Crystals of the recombinant thiol aminopeptidase PepC, from Lactoccocus lactis, have been obtained using the hanging-drop method of vapor diffusion from ammonium sulfate solutions. Crystals are rhombohedral, the space group is R32, a = 175.2 A, c = 94.5 A (hexagonal setting). The asymmetric unit probably contains one monomer of a hexameric molecule-arrangement of 300 kDa which exhibits the crystallographic point group of symmetry 32. The crystals diffract to at least 3 A resolution.

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin.

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

Structure of influenza virus haemagglutinin complexed with a neutralizing antibody.

Haemagglutinin (HA) is the influenza surface glycoprotein that interacts with infectivity-neutralizing antibodies. As a consequence of this immune pressure, it is the variable virus component, which is important in antigenic drift, that results in recurrent epidemics of influenza. We have determined the crystallographic structure of a complex formed between the antigen-binding fragment (Fab) of a neutralizing antibody and the membrane-distal domain ('HA top') of a HA subunit prepared from HA in its membrane-fusion-active conformation. A dramatic change is seen in the structure of the Fab-combining site on complex formation. Our results indicate that neutralization of infectivity by this antibody involves the inhibition of receptor binding, and demonstrate how influenza virus can maintain its conserved receptor-binding site despite the immune selective pressure for change in this region of the molecule; they also contribute to a complete description of the endosomal pH-induced fusion-active HA structure.