An Epidemiologically Significant Epitope of a 1998 Human Influenza Virus Neuraminidase Forms a Highly Hydrated Interface in the NA–Antibody Complex Lalitha Venkatramani, Elena Bochkareva, Janis T. Lee, Upma Gulati W. Graeme Laver, Alexey Bochkarev and Gillian M. Air * Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center, Oklahoma City OK 73190, USA The crystal structure ofthe complex between neuraminidase (NA) of influenza virus A/Memphis/31/98 (H3N2) and Fab of monoclonal antibody Mem5 has been determined at 2.1 A ˚ resolution and shows a novel pattern of interactions compared to other NA-Fab structures. The interface buriesa large area of 2400 A ˚ 2 and the surfaces have high complementarity. However, the interface is also highly hydrated. There are 33 water molecules in the interface R95% buried from bulk solvent, but only 13 of these are isolated from other water molecules. The rest are involved in an intricate network of water-mediated hydrogen bonds throughout the interface, stabilizing the complex. Glu199 on NA, the most criticalside-chain to the interaction as previously determined by escape mutant analysis and site-directed mutation, is located in a non-aqueous island. Glu199 and three other residues that contribute the major part of the antigen buried surface of the complex have mutated in human influenza viruses isolated after 1998, confirming that Mem5 identifies an epidemio- logically important antigenic site. We conclude that antibody selection of NA variants is a significant component of recent antigenic drift in human H3N2 influenza viruses, supporting the idea that influenza vaccines should contain NA in addition to hemagglutinin. q 2005 Elsevier Ltd. All rights reserved. Keywords: neuraminidase–Fab structure; influenza virus;antigenic drift; water-mediated hydrogen bonds *Corresponding author Introduction Influenza virus, an Orthomyxovirus, has a negative-stranded RNA segmented genome that contains coding information for about ten proteins. Influenza type A viruses are further classified into antigenic subtypes ofHA (H1 to H16) and NA (N1 to N9). Epidemic human viruses have so far only included H1, H2, H3, N1 and N2 antigens, but all subtypes have been isolated from avian species. Humans have recently been lethally infected with viruses containing H5 or H7 HA and there is considerableconcern that these or other avian subtypes could appear in human viruses and cause a major pandemic. The two major surface glycoproteins of the virus, neuraminidase (NA) and hemagglutinin (HA),are embedded in the lipid membrane of the virus and form spikes on the outer surface.Neutralizing antibodies raised by either vaccination or infection are directed to the native forms of these surface glycoproteins, and bound antibody inhibits their functions. The HA is a trimer,and it binds to sialic acid receptors on the cell surface.The NA is a tetramer of identical subunits of50,000 M r . NA cleaves terminalsialic acid from glycoconjugates such as those on the viral glycoproteins and the surface of target cells in the respiratory tract. 1,2 NA is thus a receptor-destroy- ing enzyme, removing sialic acid from carbo- hydrate chains attached to HA and NA. If NA 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Present addresses: E. Bochkareva and A. Bochkarev, Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5G 1L6, Canada; G. Laver, 3047 Barton Highway, Murrumbateman, NSW 2582, Australia. Abbreviations used: HA, haemagglutinin; NA, neuraminidase; CDR, complementarity-determining region. E-mail address of the corresponding author: gillian-air@ouhsc.edu doi:10.1016/j.jmb.2005.11.061 J. Mol. Biol. (2006) 356, 651–663