Rational and computational design of stabilized variants of cyanovirin-N that retain affinity and specificity for glycan ligands.

The 11-kDa lectin Cyanovirin-N (CVN), originally isolated from the freshwater cyanobacterium Nostoc ellipsosporum, has generated interest as a potential anti-viral agent. Therapeutic interest in CVN stems from its ability to potently and irreversibly inactivate both laboratory-adapted and naturally-occurring strains of the Human Immunodeficiency Virus (HIV) (1). This irreversible inhibition involves tight binding of CVN to the high-mannose oligosaccharides on the viral envelope glycoprotein gp120 (2). The binding event affects the flexibility of gp120, and is thought to hinder the conformational changes which are required for proper interactions with the cell-membrane receptor CD4 and co-receptors CCR5/CXCR4, which are essential for subsequent gp41-mediated membrane fusion (3, 4). The presence of glycans on gp120 is crucial to the ability of HIV to evade detection by the immune system. The dense glycan coating of gp120 gives rise to what has been termed a “glycan shield” due to its masking of the underlying immunogenic protein epitopes (5). In fact, most of the surface of the proteins exposed on the extraviral side of the envelope is covered by carbohydrates. CVN, along with a number of other lectins, represents an example of a novel class of therapeutic carbohydrate-binding agents against enveloped viruses. The antiviral activity of such molecules is dual in mechanism; first, they are able to bind to the glycans of the viral envelope and block virus entry, and second, long-term exposure to such agents leads to a progressive deletion of the glycosylation sites on the viral surface as an adaptive response to antiviral pressure. In the case of HIV, the deletion of the “glycan shield” is thought to reveal previously-obscured epitopes and allow enhanced detection and neutralization of the virus by the immune system (3, 6). CVN has also been shown to inhibit transmission by several other enveloped viruses; in addition to HIV, CVN inhibits Ebola and herpes simplex viral infection by binding to their respective envelope glycoproteins (7, 8), and influenza infection by binding to hemagglutinin (9). Wild-type CVN possesses modest stability and has a tendency to form domain-swapped dimers (10). These factors complicate its clinical use and can make biophysical and biochemical studies of the protein difficult. The most promising clinical application of CVN is as a topical microbicide, with hope of reducing HIV transmission in sub-Saharan Africa. It must thus be stable under a wide range of conditions and possess a long shelf life under harsh conditions. The interest in developing CVN as a microbicide has led to investigations of large-scale production of the protein using bacterial, yeast, and plant expression systems (11–13). Stabilization of CVN has implications for recombinant production of the protein as a therapeutic, and can aid in recombinant protein purification. In addition, availability of stabilized CVN variants facilitates studies of mutations which significantly disturb the stability of the protein fold, such as previously-characterized binding-site knockouts (14–18) or designed protein oligomers (19). We set out to design variants of CVN which are more stable to chemical and thermal unfolding, but retain the full biological activity of the wild-type protein. Here, we present CVN variants which are more stable than the wild-type protein, yet retain the native fold and carbohydrate specificity. The designed homologues represent a new background which is amenable to binding-site redesign or engineering, due to their increased tolerance of higher temperatures and denaturant concentrations compared to wild-type CVN. Protein stabilization has previously been achieved using a variety of approaches: improving core packing (20), removal of buried polar side chains or unsatisfied salt bridges (21, 22), homology-based strategies (23), mutation of charged surface residues (24), introduction of new disulfide bonds (25), turn redesign (26–28), modulation of unfolded state entropy (29), and rational considerations of unfolded state interactions (30). Often, the substitutions increase stability via a mixture of effects, such as optimized core packing and increased burial of hydrophobic surface area, which may be difficult to deconvolve (31). In this work, we employed a rational design strategy which removes buried polar groups and improves the packing within the protein core to yield increased stability. We found that kinetic denaturation by guanidine hydrochloride (GuaHCl) is very slow, and that CVN folding is not two-state. Previous studies of CVN folding thermodynamics were misled by the slow unfolding, and we provide updated thermodynamic parameters for CVN. Such data is essential for a description of CVN's complicated free energy landscape.