A Monovalent Mutant of Cyanovirin-N Provides Insight into the Role of Multiple Interactions with gp120 for Antiviral Activity^,

Abstract

Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.

Structure and Glycan Binding of a New Cyanovirin-N Homolog

“…This relation, however, cannot become crystallographic by any alternative unit cell/symmetry definition. The current structure of Cyt-CVNH is similar to previously determined crystal structures of monomeric, nondomain-swapped CV-N variants (22,23), in contrast to the domain-swapped wild type CV-N structures (13,24,25). We ascertained that the x-ray data were incompatible with a domain-swapped dimer structure by omitting the hinge-loop region (Trp 49 -Asn 53 ) in the model employed for molecular replacement.…”

A Glycoconjugate Antigen Based on the Recognition Motif of a Broadly Neutralizing Human Immunodeficiency Virus Antibody, 2G12, Is Immunogenic but Elicits Antibodies Unable To Bind to the Self Glycans of gp120

“…Perhaps Abs with these antigen binding sites readily recognize synthetic high-mannose but not natural high-mannose structures because the linker-dependent presentations described above are also adopted on the printed covalent glycan array. In addition, increased positional fluctuations may disrupt the hydrogen bonding network between the antennae of branched synthetic oligomannosides (e.g., Man 8 and Man 9 ), also facilitating Ab binding (26).…”

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

“…Numerous studies have shown, however, that both sites are necessary for viral neutralization and that destruction of either site renders the CV-N variant inactive (16,17). However, a recent study showed that in the context of a CV-N dimer that was covalently crosslinked using disulfide bonds, two out of the four possible binding sites are sufficient to maintain neutralization activity, indicating that it is the number and not the identity of sites that is important for neutralization (18,19).…”