Probing the Sialic Acid Binding Site of the Hemagglutinin-Neuraminidase of Newcastle Disease Virus: Identification of Key Amino Acids Involved in Cell Binding, Catalysis, and Fusion (original) (raw)
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Journal of Virology, 2004
The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein responsible for attachment to receptors containing sialic acid, neuraminidase (NA) activity, and the promotion of membrane fusion, which is induced by the fusion protein. Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN protein revealed the presence of a large pocket, which mediates both receptor binding and NA activities. Recently, a second sialic acid binding site on HN was revealed by cocrystallization of the HN with a thiosialoside Neu5Ac-2-S-␣(2,6)Gal1OMe, suggesting that NDV HN contains an additional sialic acid binding site. To evaluate the role of the second binding site on the life cycle of NDV, we rescued mutant viruses whose HNs were mutated at Arg516, a key residue that is involved in the second binding site. Loss of the second binding site on mutant HNs was confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to block the NA active site. Characterization of the biological activities of HN showed that the mutation at Arg516 had no effect on NA activity. However, the fusion promotion activity of HN was substantially reduced by the mutation. Furthermore, the mutations at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest that the second binding site facilitates virus infection and growth by enhancing the fusion promotion activity of the HN.
Journal of General Virology, 2004
Mutations were generated in residues at the putative catalytic site of the haemagglutinin-neuraminidase (HN) protein of Newcastle disease virus Clone 30 strain (Arg498, Glu258, Tyr262, Tyr317 and Ser418) and their effects on its three associated activities were studied. Expression of the mutant proteins at the surface of HeLa cells was similar to that of the wild-type. Sialidase, receptor-binding and fusion-promotion activities were affected to different degrees for all mutants studied. Mutant Arg498Lys lost most of its sialidase activity, although it retained most of the receptor-binding activity, suggesting that, for the former activity, besides the presence of a basic residue, the proximity to the substrate molecule is also important, as Lys is shorter than Arg. Proximity also seems to be important in substrate recognition, since Tyr262Phe retained most of its sialidase activity while Tyr262Ser lost most of it. Also, Ser418Ala displayed most of the wild-type sialidase activity. However, a kinetic and thermodynamic study of the sialidase activity of the Tyr262Ser and Ser418Ala mutants was performed and revealed that the hydroxyl group of these residues also plays an important role in catalysis, since such activity was much less effective than that of the wild-type and these mutations modified their activation energy for sialidase catalysis. The discrepancy of the modifications in sialidase and receptor-binding activities in the mutants analysed does not account for the topological coincidence of the two sites. These results also suggest that the globular head of HN protein may play a role in fusion-promotion activity.
Structure, 2005
In addition to receptor binding and NA activity, HN Robert A. Lamb, 1,2 and Theodore S. Jardetzky 1, * has a fusion-promoting activity that plays an important 1 Department of Biochemistry, Molecular Biology, role in virus-cell fusion. The coexpression of HN and F and Cell Biology derived from the same paramyxovirus lowers the acti-2 Howard Hughes Medical Institute vation energy barrier for triggering F protein-mediated Northwestern University fusion (Russell et al., 2001). Coimmunoprecipitation as-Evanston, Illinois 60208-3500 says suggest that F and HN exist in a complex, and a great deal of effort has been spent to map the regions of F and HN that interact. Mutations have been iden-Summary tified in several domains of HN that decrease or abolish fusogenic activity with no effect on receptor recognition The paramyxovirus hemagglutinin-neuraminidase (HN) (reviewed in Colman and Lawrence, 2003). However, a functions in virus attachment to cells, cleavage of siaconsensus view of the domains of interaction between lic acid from oligosaccharides, and stimulating mem-HN and F has not been obtained to date. brane fusion during virus entry into cells. The struc-HN is a type II integral membrane protein that spans tural basis for these diverse functions remains to be the membrane once and contains an N-terminal cytofully understood. We report the crystal structures of plasmic tail, a single N-terminal transmembrane (TM) the parainfluenza virus 5 (SV5) HN and its complexes domain, a membrane-proximal stalk domain, and a with sialic acid, the inhibitor DANA, and the receptor large C-terminal globular head domain (Hiebert et al., sialyllactose. SV5 HN shares common structural fea-1985). The globular head domain contains the receptor tures with HN of Newcastle disease virus (NDV) and binding and enzymatic activity (Parks and Lamb, 1990; human parainfluenza 3 (HPIV3), but unlike the pre-Scheid et al., 1972; Thompson and Portner, 1987). HN viously determined HN structures, the SV5 HN forms is glycosylated and noncovalently associated to form a a tetramer in solution, which is thought to be the tetramer, based on biochemical, crosslinking, and physiological oligomer. The sialyllactose complex reelectron microscopy studies, which, depending on the veals intact receptor within the active site, but no mavirus, can be composed of two disulfide-linked dimers jor conformational changes in the protein. The SV5 (McGinnes et al., 1993; Ng et al., 1989, 1990; Thompson HN structures do not support previously proposed et al., 1988). The covalent linkage occurs through a cysmodels for HN action in membrane fusion and sugteine residue in the stalk domain. The specific residues gest alternative mechanisms by which HN may proinvolved in tetramer association have not been idenmote virus entry into cells. tified but have been suggested to reside in the TM domain, the cytoplasmic domain, the extracellular domain, or in all three domains (Parks and Lamb, 1990;
Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase
Nature structural biology, 2000
Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a ...
Journal of Virology, 2002
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.
Proceedings of the National Academy of Sciences, 2011
The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcast...
Journal of Virology, 2003
The tetrameric paramyxovirus hemagglutinin-neuraminidase (HN) protein mediates attachment to sialic acid-containing receptors as well as cleavage of the same moiety via its neuraminidase (NA) activity. The X-ray crystallographic structure of an HN dimer from Newcastle disease virus (NDV) suggests that a single site in two different conformations mediates both of these activities. This conformational change is predicted to involve an alteration in the association between monomers in each HN dimer and to be part of a series of changes in the structure of HN that link its recognition of receptors to the activation of the other viral surface glycoprotein, the fusion protein. To explore the importance of the dimer interface to HN function, we performed a site-directed mutational analysis of residues in a domain defined by residues 218 to 226 at the most membrane-proximal part of the dimer interface in the globular head. Proteins carrying substitutions for residues F220, S222, and L224 in this domain were fusion deficient. However, this fusion deficiency was not due to a direct effect of the mutations on fusion. Rather, the fusion defect was due to a severely impaired ability to mediate receptor recognition at 37°C, a phenotype that is not attributable to a change in NA activity. Since each of these mutated proteins efficiently mediated attachment in the cold, it was also not due to an inherent inability of the mutated proteins to recognize receptors. Instead, the interface mutations acted by weakening the interaction between HN and its receptor(s). The phenotype of these mutants correlates with the disruption of intermonomer subunit interactions.
Journal of Virology, 2004
The Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein mediates attachment to cellular receptors. The fusion (F) protein promotes viral entry and spread. However, fusion is dependent on a virus-specific interaction between the two proteins that can be detected at the cell surface by a coimmunoprecipitation assay. A point mutation of I175E in the neuraminidase (NA) active site converts the HN of the Australia-Victoria isolate of the virus to a form that can interact with the F protein despite negligible receptor recognition and fusion-promoting activities. Thus, I175E-HN could represent a fusion intermediate in which HN and F are associated and primed for the promotion of fusion. Both the attachment and fusion-promoting activities of this mutant HN protein can be rescued either by NA activity contributed by another HN protein or by a set of four substitutions at the dimer interface. These substitutions were identified by the evaluation of chimeras composed of segm...