Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and x-ray crystallography

Abstract

The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.

Influenza virus binds its host cell using multiple dynamic interactions

“…S7), which agrees with the observations made during the AFM virus-cell pulling experiments. From NMR studies with purified ligands (20), the dissociation constant of X-31 HA to sialyllactose was reported to be 2.1 mM (2,6 linked) and 3.2 mM (2,3 linked). We suppose that these rather small differences observed using purified binding partners may not become obvious under conditions mimicking more closely the biological situation, as in our study.…”

“…The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al (1992) Biochemistry 31:9609-9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches.…”

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses

“…Characterization of A2-PC-QDs was performed by 5 direct MALDI-TOFMS (Nagahori et al, 2009;Ohyanagi et al, 2011) and the results showed the advent of an expected molecular ion signal at m/z 3155.7 corresponding to the heterodisulfide A2-S-S-PC instead of disappearance of the signal at m/z 908.7 due to ao-S-S-PC corresponding to free aminooxy-functional groups. As standards or simple model compounds in the competitive binding assay, 2-aminobenzamide (2AB) labeled A2 (2AB-A2, 1) (Dell et al, 2008), and two known sialosides, methyl 5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosonic acid (2) (Sauter et al, 1992) and octyl 5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosonic acid (3) (Crich and Li, 2007), were also used.…”

“…3). To test the feasibility of this method for screening HA blockers as candidates for anti-influenza drugs, we employed preliminarily two known simple derivatives of sialic acid, methyl sialoside (2), which is already known to bind with the BroHA of X-31 strain (Sauter et al, 1992), and octyl sialoside (3). Each compound was added to a mixture of A2-PC-QDs and BroHA in the concentration range designated for the quantitative FP assay.…”

Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand

“…Because of the dramatic effect of the 25 mM soak on the native crystals, it was decided to proceed with cocrystallization at this concentration. This choice of concentration was also supported by the known dissociation constant of 2.3 sialyllactose from influenza hemagglutinin (Kd = 3.2 mM; Sauter et al, 1992). which has similar affinity and binding specificity to sialoadhesin (Crocker et al, 1991).…”