A SINGLE SEQUENCE CHANGE DESTABILIZES THE INFLUENZA-VIRUS NEURAMINIDASE TETRAMER

A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-N eu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, wh ich sits in a pocket in the bottom of the active site of the enzyme, a nd the 4-guanidinium moiety of the inhibitor to be lost. NA ''heads'' of the resistant enzyme produced only a few small crystals under condi tions in which the wild-type enzyme readily formed large crystals. The se small crystals were of sufficient quality to yield X-ray crystallog raphic data which confirmed the E119G change and demonstrated the pres ence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activit y per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetrame rs, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crysta ls obtained were probably from the few tetramers remaining intact in t he preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examinati on of the known X-ray crystallographic structure of the wild-type NA r eveals a salt bridge between Glu119 and Arg156 of the same monomer. Ar g156 is a conserved amino acid that is situated at the interface betwe en monomers, and a salt link between this amino acid and Glu119 may co ntribute to the stability of enzyme tetramers. It is suggested that th e E119G alteration in the 4-GuDANA-resistant NA leads to the abrogatio n of this interaction and thus to the instability of the NA tetramers. (C) 1997 Academic Press.