The proteins were tested against a total of 33 strains from three clades. neutralizing anti-HIV antibodies. The improvement in activity and broad cross-strain HIV neutralization exhibited by these molecules holds promise for the future therapeutic utility of these and other engineered CV-N variants. Keywords:crystal structure, domain-swapped dimer, protein engineering Cyanovirin-N (CV-N), a cyanobacterial lectin, is uniquely positioned to become a therapeutic and prophylactic for diseases caused by enveloped viruses. CV-N is a small, two-domain protein that neutralizes HIV by specifically binding to high mannose glycans around the envelope glycoprotein gp120, thereby preventing interaction of the computer virus with a host cell (1,2). In addition to its potent activity against HIV, CV-N is also active against a number of other enveloped viruses including influenza (3,4), Ebola (5,6), hepatitis C (7), and herpesvirus 6 (2). The two domains of CV-N are homologous in both their sequence [32% sequence identity and 58% sequence similarity (8)] and their three-dimensional structure (9,10). Wild-type (WT) CV-N exists mainly as a monomer in solution and a domain-swapped dimer in crystals (Fig. S1). NMR structures of the monomer show that the protein is an ellipsoid with ten -strands and four 310-helical turns, approximately 55 in length and 25 wide (Fig. S1A). Each CV-N monomer contains two symmetrically related structural domains (A and B), with each domain containing a carbohydrate binding site that specifically interacts with (1-2) linked oligomannose moieties within Man-8 or Man-9 glycans (9,1113). Domain A contains the N and C termini and includes residues 139 and 90101, and domain B contains residues 4089. Although purified as a monomer, a trapped, metastable domain-swapped dimer can be formed during folding and crystallization, and WT CV-N crystallizes exclusively as a domain-swapped dimer (10,14,15). In the domain-swapped dimer structure, domain A interacts with B to form a monomer-like unit, whereas domain A and B interact to form the second monomer-like unit (Fig. S1BandC). The domain swapping does not result in additional intramolecular interactions, but instead results from the extension of residues 50 to 53 across the interface. Carbohydrate binding is similar in the domain-swapped crystal structures and monomeric NMR structures, although binding in the B and B domains is not seen in crystal structures due to potential steric constraints or crystal packing artifacts (15) (Fig. S1AandB). The two binding sites in monomeric CV-N exhibit distinct affinities for carbohydrate in solution: the binding site in domain B, located distal from the N and C termini, has an equilibrium dissociation constant (KD) of approximately 140 nM for Man1 2Man disaccharide, which is about 10-fold higher affinity than the binding site in domain A, located near the termini, which binds to Man1 2Man disaccharide with aKDof about 1.5 M (9). 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 Araloside V 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 RaLP that is important for neutralization (18,19). These results point toward a key role for avidity in the viral neutralization activity of CV-N. A number of groups have attempted to study the oligomerization of CV-N to determine whether the domain swapping is Araloside V a crystallization artifact or a biologically relevant state. However, because the domain-swapped dimer of WT CV-N is not stable at physiological temperatures, a significant amount of purified dimer may revert to monomer during the course of a viral neutralization assay (14). Therefore, mutations have been used to stabilize either the monomer (14) or the domain-swapped dimer (14,20,21). The effect of dimerization remains unclear, as some groups have concluded that the dimeric state is more active than monomeric WT CV-N (21), whereas others find that monomeric and dimeric variants have Araloside V similar antiviral activities (20). In this study, we show that by linking two CV-N molecules together in a head-to-tail fashion, we Araloside V can stabilize the domain-swapped dimeric form of the protein in solution. These linked dimers show enhanced HIV neutralization compared to WT CV-N against 33 strains from 3 clades. In addition, we show that although two carbohydrate binding sites are sufficient for activity as previously reported (18,19), variants with more binding sites (three or four) have increased neutralization activity. == Results == == Design and Construction of CV-N Oligomers. == To directly assay the.