Molecular Mechanisms of Inhibition of Influenza by Surfactant Protein D Revealed by Large-Scale Molecular Dynamics Simulation

Abstract

Surfactant protein D (SP-D), a mammalian C-type lectin, is the primary innate inhibitor of influenza A virus (IAV) in the lung. SP-D interactions with highly branched viral N-linked glycans on hemagglutinin (HA), an abundant IAV envelope protein and critical virulence factor, promote viral aggregation and neutralization through as yet unknown molecular mechanisms. Two truncated human SP-D forms, wild-type (WT) and double mutant D325A+R343V, representing neck and carbohydrate recognition domains are compared in this study. Whereas both WT and D325A+R343V bind to isolated glycosylated HA, WT does not inhibit IAV in neutralization assays; in contrast, D325A+R343V neutralization compares well with full-length native SP-D. To elucidate the mechanism for these biochemical observations, we have solved crystal structures of D325A+R343V in the presence and absence of a viral nonamannoside (Man9). Based on the D325A+R343V/Man9 structure and other crystallographic data, models of complexes between HA and WT or D325A+R343V were produced and subjected to molecular dynamics. Simulations reveal that whereas WT and D325A+R343V both block the sialic acid receptor site of HA, the D325A+R343V complex is more stable, with stronger binding due to additional hydrogen bonds and hydrophobic interactions with HA residues. Furthermore, the blocking mechanism of HA differs for WT and D325A+R343V due to alternate glycan binding modes. The combined results suggest a mechanism through which the mode of SP-D/HA interaction could significantly influence viral aggregation and neutralization. These studies provide the first atomic-level molecular view of an innate host defense lectin inhibiting its viral glycoprotein target.

Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

“…We recently reported that D325AϩR343V had increased neutralizing activity of Aichi68 compared with native SP-D or SP-A (8). Using molecular dynamic simulations, we showed that this increased neutralizing activity relates to an increased ability of D325AϩR343V to occlude the sialic acid binding site of the Aichi68 HA compared with wild-type NCRD (8). In addition, these and prior studies indicated an increased ability of the R343V and D325AϩR343V to crosslink HA molecules on the basis of its distinctive binding orientation to the HA after attaching to glycan 165 (5, 7).…”

“…Again, wild-type NCRD did not inhibit this strain. We recently reported that D325Aϩ R343V has significantly greater neutralizing activity for Aichi68 compared with native SP-D dodecamers, native SP-A, or R343V and that wild-type SP-D NCRD does not inhibit this strain (8). As shown in Fig.…”

“…We have reported, however, that mutating residues surrounding the lectin site of the SP-D NCRD confers increased viral binding and neutralizing activity. In particular, a mutant form of the trimeric carbohydrate recognition domain (i.e., NCRD) of SP-D with combinatorial substitutions at the 325 and 343 positions (D325AϩR343V) has greatly increased activity against seasonal IAV in vitro and in vivo (8).…”

Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions

“…Typical hemagglutination inhibition assays are performed using serum to detect the presence and levels of antibodies against HA. Because SP-D prevents IAV interaction with host cells, it can be used to inhibit hemagglutination activity, in a dose-dependent manner(15, 20, 54). …”

“…The wild-type recombinant human NCRD (huNCRD) has been shown to bind glycans but lacks antiviral activity because of absence of cooperative binding effect from the multiple heads (49–51). Gain-of-function mutants R343V and double mutant D325A+R343V (D+R), which have been shown to possess increased virus binding and neutralizing activity (48, 49, 52–54), were also used in bioassays. Mutant names are indicative of amino acid substitutions flanking the lectin site in the huNCRD protein.…”