The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid.

Abstract

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R‐factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co‐ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.

Influenza neuraminidase

“…Early X-ray crystal structures of NA heads complexed with sialic acid were solved for N2 22 , N9 2324 and type B 25 NAs. These showed a conserved 6-bladed propeller structure, each blade made up of four anti-parallel beta sheets stabilized by disulfide bonds and connected by loops of variable length.…”

“…The active site is highly conserved in spatial as well as sequence properties, and showed little change in conformation on binding a transition state analogue inhibitor (2-deoxy-2,3-dehydro-N-acetylneuraminic acid, DANA) or the sialic acid product of the reaction. Eleven conserved amino acids make contact with DANA (Figure 1) while another 6 conserved amino acids form a “second shell” 25 that holds the active site residues in place. The whole appears rather rigid, providing the basis of several drug design approaches (see reviews 2,6,26–30 .…”

“…Ca 2+ is required for activity and stability 4344 , and up to five Ca 2+ ions per tetramer are seen in the crystal structures. One Ca 2+ sits at the four-fold axis and so may stabilize the tetramer, although it is coordinated via water molecules and its affinity is weak 25 . It is not seen in several structures, either due to low resolution or low affinity.…”

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

“…Then, the sequence of human sialidase was aligned with the sequences of SCRs manually. A backbone of human sialidase corresponding to each SCR was built by fitting a fragment from one of the homologues, and a rule-based procedure was used to determine the sidechain conformations (Sutcliffe et al 1987;Burmeister et al 1992). Loop regions were constructed with fragments selected from the protein substructure database (Jones and Thirup 1986;Claessens et al 1989).…”

Diversidade genética da neuraminidase de vírus Influenza A, isolados de crianças internadas na cidade de São Paulo, de 1995 a 2006.

“…A estrutura da cabeça da NA subtipo 2 (N2) é um homotetrâmero, no qual cada monômero é composto de seis folhas beta, topologicamente idênticas, dispostas em formato de hélice. Os resíduos de açúcar estão ligados a quatro dos cinco sítios potenciais de glicosilação da cabeça (BURMEISTER, RUIGROK, CUSACK, 1992).…”