The presence of the additional carbohydrate-binding domain in the 68 kDa form of the bacterial sialidase reported here is a further example of a combination of carbohydrate binding and cleaving domains which we observed in the sialidase from Vibrio cholerae. This dual function may be common, but only to other bacterial and parasitic sialidases, but also to other secreted glycosidases involved in pathogenesis. The bacterium may have acquired both the immunoglobulin module and the galactose-binding module from eukaryotes, as the enzyme shows a remarkable similarity to a fungal galactose oxidase which possesses similar domains performing different functions and assembled in a different order.

Sialidases (EC 3.2

The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.

Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth ␤ sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.

We recently reported the first crystal structure of a paramyxovirus hemagglutinin-neuraminidase (HN) from Newcastle disease virus. This multifunctional protein is responsible for binding to cellular sialyl-glycoconjugate receptors, promotion of fusion through interaction with the second viral surface fusion (F) glycoprotein, and processing progeny virions by removal of sialic acid from newly synthesized viral coat proteins. Our structural studies suggest that HN possesses a single sialic acid recognition site that can be switched between being a binding site and a catalytic site. Here we examine the effect of mutation of several conserved amino acids around the binding site on the hemagglutination, neuraminidase, and fusion functions of HN. Most mutations around the binding site result in loss of neuraminidase activity, whereas the effect on receptor binding is more variable. Residues E401, R416, and Y526 appear to be key for receptor binding. The increase in fusion promotion seen in some mutants that lack receptor binding activity presents a conundrum. We propose that in these cases HN may be switched into a fusion-promoting state through a series of conformational changes that propagate from the sialic acid binding site through to the HN dimer interface. These results further support the single-site model and suggest certain residues to be important for the triggering of fusion.Viruses belonging to the family Paramyxoviridae are major causative agents for respiratory illnesses in humans, particularly in children. Members of the Paramyxovirinae subfamily include the human parainfluenza viruses (PIVs), mumps viruses, Newcastle disease virus (NDV), Sendai virus, and simian virus 5. Infection of host cells by paramyxoviruses is accomplished by the interaction of two surface glycoproteins, hemagglutinin-neuraminidase (HN) and the fusion (F) protein. HN possesses both the receptor recognition of sialic acid at the termini of host glycoconjugates and neuraminidase activity to hydrolyze sialic acid from progeny virion particles to prevent viral self-aggregation (14,23,24). In addition to these activities, HN has been shown to promote fusion through its interaction with the F protein, which involves residues from the stalk and the globular head region of HN (1,2,8,26,31,34), thereby allowing the entry of viral RNA.Recently, we determined the first crystal structure of the globular head region of the Newcastle disease virus HN (6). HN displays the six-bladed ␤-propeller fold typical of other sialidases/neuraminidases, whose structures are known (5, 7, 9, 35). Two crystal forms of the dimeric HN molecule were determined: a pH 6.5 hexagonal crystal form that would only grow in the presence of the inhibitor 2-deoxy-2,3-dehydro-Nacetyl-neuraminic acid (Neu5Ac2en) and a pH 4.6 orthorhombic crystal form grown in the absence of ligand but into which ligands could be soaked. The association of monomers in these two forms is very different, suggesting a flexible interface between the monomers that may have functional significanc...