Engineering an Obligate Domain-Swapped Dimer of Cyanovirin-N with Enhanced Anti-HIV Activity Brendan S. Kelley, Leng Chee Chang, and Carole A. Bewley* Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0820 Received January 7, 2002 Three-dimensional (3D) domain swapping refers to the event wherein one protein molecule exchanges a structural domain with the same of a second identical molecule to form an intertwined dimer or oligomer. 1 Structural domains that have been observed to undergo domain swapping range from single -strands, 2a R-helices, 2b and loops, 2c to larger structural domains. 1,2d,e Domain exchange occurs about a so-called “hinge-loop”, or linker, which separates the two regions of each monomer of an intertwined dimer. Thus, the 3D structures of each half of a domain-swapped dimer are nearly identical to the parent monomer, with the exception of the linker which becomes extended to accommodate domain exchange. The anti-HIV cyanobacterial protein cyanovirin-N (CVN) can undergo domain-swapping under nonphysiological conditions, 2e demonstrated structurally by NMR 3a,b and X-ray crystallography. 2e Specifically, following purification by reversed-phase HPLC at low pH in the presence of organic solvents and lyopholization, the resolubilized protein is present as a mixture of approximately 70: 25:5 monomer:dimer:oligomer (Supporting Information). However, the domain-swapped dimeric form is not favored at neutral pH and converts back into a monomer upon titration to pH > 5.0 to yield samples comprising only 5-10% dimeric CVN. 2e Interest in CVN arises from its ability (at nM concentrations) to potently block viral entry by human immunodeficiency viruses (HIV) through highly avid interactions with the viral envelope glycoprotein gp120. 4a,b CVN:gp120 interactions are governed by high affinity binding of CVN to the D1 and D3 arms of oligomannose-8 (Man 8 ) D1D3 and oligomannose-9 (Man 9 ), 5 mam- malian oligosaccharides that are abundant on the viral surface. This unprecedented specificity arises from the presence of two extensive carbohydrate binding pockets that are specific for the disaccharide ManR(1-2)ManR, which represents the termini of the more accessible D1 and D3 arms of Man 8 and Man 9 . 6 While structural studies of domain-swapped dimers are fairly plentiful, corresponding functional studies are lacking. This may be attributed to the fact that the domain-swapped dimeric or oligomeric forms of various proteins observed in the crystal lattice are insufficiently stable at low concentrations to study function. 7 Given that CVN blocks viral entry through carbohydrate-mediated interactions 5,6,8 and that increasing valency in protein-carbohydrate interactions is known to decrease apparent equilibrium dissociation (K D ) constants, 9 we supposed that the domain-swapped dimeric form of CVN, which possesses four carbohydrate binding sites, should be twice as potent as the naturally occurring monomer which possesses two carbohydrate binding sites. 5,6 To test this hyposthesis rigorously, 10 we have engineered an obligate domain-swapped dimeric form of CVN as described below. CVN has a pseudosymmetrical 3D structure comprising two adjacent triple-stranded antiparallel -sheets in the back of the protein (as viewed in Figure 1a), and two oppositely placed -hairpins on the front of the protein, each of which is preceded by a single 3 10 helical turn. 11 The homologous sequence repeats (residues 1-50 and 51-101) are separated by a central linker (comprising Gln50-Pro51-Ser52-Asn53) that precisely crosses over -strand 4, 11 and facilitates domain swapping. 2e On the basis of this structure, we predicted that a mutant bearing a shortened hinge linker would be restricted to form a domain-swapped dimer due to unfavorable steric interactions that would occur between the shortened linker and the underlying -hairpin if the monomer fold was assumed. Since the presence of proline in hinge linkers correlates with domain-swapping 12 and since Ser52 and Asn 53 participate in carbohydrate binding, 6 we chose to delete Gln50 from the linker when engineering an obligate dimer. The CVN Gln50 deletion mutant (ΔQ50-CVN) was constructed by site-directed mutagenesis (Supporting Information), and uni- formly labeled 15 N-ΔQ50-CVN was overexpressed as described previously. 11 The recombinant protein was purified from a crude cell lysate (50% aq CH 3 CN) in a single step by reversed-phase HPLC. The presence and relative abundance of monomeric and dimeric wildtype CVN (wtCVN) can be readily assessed from a 1 H- 15 N single quantum coherence correlation spectrum (HSQC) which shows doubling of 18 resolved signals. 3a 1 H- 15 N HSQC spectra of NMR samples of ΔQ50-CVN (10% D 2 O) prepared with and without adjusting the pH (measured pH values of 6.4 and 2.3, * To whom correspondence should be addressed. E-mail: cb194k@nih.gov. Figure 1. Solution structures of (a) monomeric wtCVN in complex with 2 equiv of the disaccharide ManR(1-2)ManR (PDB accession code 1IIY 6 ) and (b) the domain-swapped wtCVN dimer (1J4V). 3b In (a), the two sequential domains are colored red and blue, respectively; the expansion highlights the hinge-linker about which domain-swapping occurs. In (b) the two intertwined monomers appear as red and blue ribbons. The figure was generated using the program MolMol. 18 Published on Web 03/12/2002 3210 9 J. AM. CHEM. SOC. 2002, 124, 3210-3211 10.1021/ja025537m Not subject to U.S. copyright. Publ. 2002 Am. Chem. Soc.