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Structure and Glycan Binding of a New Cyanovirin-N Homolog*

  • Elena Matei
    Affiliations
    Department of Structural Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15260
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  • Rohan Basu
    Affiliations
    Department of Structural Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15260

    Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802
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  • William Furey
    Affiliations
    Department of Pharmacology & Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261

    Biocrystallography Laboratory, Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240
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  • Jiong Shi
    Affiliations
    Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, 37232
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  • Conor Calnan
    Affiliations
    Department of Structural Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15260

    the Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Christopher Aiken
    Affiliations
    Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, 37232
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  • Angela M. Gronenborn
    Correspondence
    To whom correspondence should be addressed: Dept. of Structural Biology, University of Pittsburgh, School of Medicine, 3501 Fifth Ave., BST3/Rm. 1050, Pittsburgh, PA 15260. Tel.: 412-648-9959; Fax: 412-648-9008
    Affiliations
    Department of Structural Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15260
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  • Author Footnotes
    * This work was supported by National Institutes of Health grant RO1GM080642 (to A. M. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental Figs. S1–S4.The atomic coordinates and structure factors (code 5K79) have been deposited in the Protein Data Bank (http://ww.pdb.org/).
Open AccessPublished:July 07, 2016DOI:https://doi.org/10.1074/jbc.M116.740415
      The HIV-1 envelope glycoprotein gp120 is heavily glycosylated and bears numerous high mannose sugars. These sugars can serve as targets for HIV-inactivating compounds, such as antibodies and lectins, which bind to the glycans and interfere with viral entry into the target cell. We determined the 1.6 Å x-ray structure of Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece7424, and elucidated its glycan specificity by NMR. The Cyt-CVNH structure and glycan recognition profile are similar to those of other CVNH proteins, with each domain specifically binding to Manα(1–2)Manα units on the D1 and D3 arms of high mannose glycans. However, in contrast to CV-N, no cross-linking and precipitation of the cross-linked species in solution was observed upon Man-9 binding, allowing, for the first time, investigation of the interaction of Man-9 with a member of the CVNH family by NMR. HIV assays showed that Cyt-CVNH is able to inhibit HIV-1 with ∼4-fold higher potency than CV-NP51G, a stabilized version of wild type CV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin for potential microbicidal use.

      Introduction

      Despite the effective use of anti-retroviral therapies as a means to treat HIV infection and prolong the lifespan of those affected by AIDS, the number of HIV infections worldwide continues to grow. Unfortunately, at present, no vaccine is available to protect against HIV, creating the need to develop safe, effective, and acceptable prevention strategies that will help halt the spread of HIV infection globally. Promising candidates for inclusion into microbicides are lectins, which are carbohydrate-binding proteins that are present in a variety of plant, fungal, and cyanobacterial species. Over the last two decades, several lectins have been identified that potently block viral infection, being active against HIV and influenza virus (
      • Boyd M.R.
      • Gustafson K.R.
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      • Nara P.L.
      • Pannell L.K.
      • et al.
      Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.
      ,
      • Mori T.
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      • Sowder 2nd, R.C.
      • Bringans S.
      • Gardella R.
      • Berg S.
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      • Turpin J.A.
      • Buckheit Jr., R.W.
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      • Boyd M.R.
      Isolation and characterization of griffithsin, a novel HIV-inactivating protein, from the red alga Griffithsia sp.
      ,
      • O'Keefe B.R.
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      • Buckheit R.
      • Boyd M.R.
      Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.
      ,
      • Koharudin L.M.
      • Gronenborn A.M.
      Antiviral lectins as potential HIV microbicides.
      ). One such example is the extensively explored cyanobacterial lectin cyanovirin-N (CV-N),
      The abbreviations used are:
      CV-N
      cyanovirin-N
      CVNH
      cyanovirin-N homolog
      ASU
      asymmetric unit
      PDB
      Protein Data Bank
      HSQC
      heteronuclear single quantum coherence.
      which possesses virucidal properties against HIV types I and II, simian immunodeficiency virus, and other enveloped viruses like Ebola and influenza at nanomolar concentrations (
      • Bewley C.A.
      • Gustafson K.R.
      • Boyd M.R.
      • Covell D.G.
      • Bax A.
      • Clore G.M.
      • Gronenborn A.M.
      Solution structure of cyanovirin-N, a potent HIV-inactivating protein.
      ,
      • Barrientos L.G.
      • Gronenborn A.M.
      The highly specific carbohydrate-binding protein cyanovirin-N: structure, anti-HIV/Ebola activity and possibilities for therapy.
      ,
      • Shenoy S.R.
      • Barrientos L.G.
      • Ratner D.M.
      • O'Keefe B.R.
      • Seeberger P.H.
      • Gronenborn A.M.
      • Boyd M.R.
      Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.
      ). Its anti-HIV activity is mediated by recognizing and interacting with high mannose glycans (Man-8, Man-9) that are present on the envelope glycoprotein gp120. Indeed, HIV-1 gp120 is highly glycosylated, and N-linked glycans account for approximately half of its molecular mass (
      • Lasky L.A.
      • Groopman J.E.
      • Fennie C.W.
      • Benz P.M.
      • Capon D.J.
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      • Nakamura G.R.
      • Nunes W.M.
      • Renz M.E.
      • Berman P.W.
      Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein.
      ,
      • Leonard C.K.
      • Spellman M.W.
      • Riddle L.
      • Harris R.J.
      • Thomas J.N.
      • Gregory T.J.
      Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.
      ). CV-N specifically binds the terminal Manα(1–2)Manα epitopes on the D1 and D3 arms of Man-8 and Man-9 glycans (
      • Bolmstedt A.J.
      • O'Keefe B.R.
      • Shenoy S.R.
      • McMahon J.B.
      • Boyd M.R.
      Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner.
      ,
      • Shenoy S.R.
      • O'Keefe B.R.
      • Bolmstedt A.J.
      • Cartner L.K.
      • Boyd M.R.
      Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins.
      ,
      • Bewley C.A.
      • Otero-Quintero S.
      The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.
      ,
      • Botos I.
      • O'Keefe B.R.
      • Shenoy S.R.
      • Cartner L.K.
      • Ratner D.M.
      • Seeberger P.H.
      • Boyd M.R.
      • Wlodawer A.
      Structures of the complexes of a potent anti-HIV protein cyanovirin-N and high mannose oligosaccharides.
      ,
      • Sandström C.
      • Berteau O.
      • Gemma E.
      • Oscarson S.
      • Kenne L.
      • Gronenborn A.M.
      Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR.
      ). The mechanism of action for mannose-binding lectins is assumed to involve the inability of the lectin-bound gp120 to productively engage the host cell CD4 and CCR5 receptors, effectively preventing the necessary conformational changes required for membrane fusion and viral entry into the host cell. At present, efforts to develop lectins such as CV-N for therapeutic use are focused on topical applications in microbicides. All known anti-HIV lectins vary in their degrees of potency and some were found to have mitogenic activity (
      • Huskens D.
      • Vermeire K.
      • Vandemeulebroucke E.
      • Balzarini J.
      • Schols D.
      Safety concerns for the potential use of cyanovirin-N as a microbicidal anti-HIV agent.
      ). Therefore, the continued search for and characterization of novel HIV inhibitory lectins are important for moving microbicide development forward (
      • Tsai C.C.
      • Emau P.
      • Jiang Y.
      • Tian B.
      • Morton W.R.
      • Gustafson K.R.
      • Boyd M.R.
      Cyanovirin-N gel as a topical micro-bicide prevents rectal transmission of SHIV89.6P in macaques.
      ,
      • Gupta S.K.
      • Nutan A.
      Clinical use of vaginal or rectally applied microbicides in patients suffering from HIV/AIDS.
      ,
      • Olsen J.S.
      • Easterhoff D.
      • Dewhurst S.
      Advances in HIV microbicide development.
      ). For CV-N, the presence of two binding sites and the multivalency of the carbohydrate have been implicated as important factors for its anti-HIV activity (
      • Barrientos L.G.
      • Gronenborn A.M.
      The highly specific carbohydrate-binding protein cyanovirin-N: structure, anti-HIV/Ebola activity and possibilities for therapy.
      ). Unfortunately, cross-linking-mediated aggregation and precipitation have hampered studies of CV-N/Man-9 interactions at the atomic level in vitro (
      • Shenoy S.R.
      • Barrientos L.G.
      • Ratner D.M.
      • O'Keefe B.R.
      • Seeberger P.H.
      • Gronenborn A.M.
      • Boyd M.R.
      Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.
      ,
      • Matei E.
      • Zheng A.
      • Furey W.
      • Rose J.
      • Aiken C.
      • Gronenborn A.M.
      Anti-HIV activity of defective cyanovirin-N mutants is restored by dimerization.
      ,
      • Barrientos L.G.
      • Matei E.
      • Lasala F.
      • Delgado R.
      • Gronenborn A.M.
      Dissecting carbohydrate-cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
      ) and have precluded unambiguous determination of whether multivalent and multisite sugar/protein interactions are a necessary prerequisite for the antiviral activity of cyanovirin-N homolog (CVNH) proteins. However, in contrast, considerable atomic level information is available on CV-N binding to substructures of Man-8 and Man-9.
      To elucidate the structural and mechanistic basis for the difference between CV-N and Cyt-CVNH, we determined the Cyt-CVNH crystal structure and assessed Man-2, Man-3, and Man-9 binding by solution NMR. The structure of Cyt-CVNH is similar to that of other members of the CVNH family, also possessing two carbohydrate-binding sites, one per domain. However, in contrast to CV-N, no cross-linking and aggregation is observed in the interaction with Man-9, permitting, for the first time, determination of accurate affinities for Man-9 binding to a CVNH lectin.
      Cyt-CVNH inhibits HIV-1 in the low nanomolar concentration range and possesses ∼4-fold higher potency than CV-N. Based on these structural and functional results, we suggest that Cyt-CVNH holds significant promise for future clinical applications.

      Results

      Crystal Structure of Cyt-CVNH

      Here, we report the crystal structure of a new CVNH, Cyt-CVNH, a recently identified lectin from the cyanobacterium Cyanothece7424 (
      • Bandyopadhyay A.
      • Elvitigala T.
      • Welsh E.
      • Stöckel J.
      • Liberton M.
      • Min H.
      • Sherman L.A.
      • Pakrasi H.B.
      Novel metabolic attributes of the genus Cyanothece, comprising a group of unicellular nitrogen-fixing cyanobacteria.
      ), which comprises two tandem sequence repeats and exhibits ∼43% identity with CV-N. Cyanothece sp. PCC 7424 is a unicellular cyanobacterium isolated from rice fields in Senegal. The genome shows that these cells have the ability to store the products of both photosynthesis (glycogen) and nitrogen fixation (cyanophycin) as intracellular inclusion bodies (
      • Bandyopadhyay A.
      • Elvitigala T.
      • Welsh E.
      • Stöckel J.
      • Liberton M.
      • Min H.
      • Sherman L.A.
      • Pakrasi H.B.
      Novel metabolic attributes of the genus Cyanothece, comprising a group of unicellular nitrogen-fixing cyanobacteria.
      ). Beyond these basic findings, however, the precise role of Cyt-CVNH within the host is unknown at present.
      The crystal structure of Cyt-CVNH was solved at 1.6 Å resolution by molecular replacement for orthorhombic crystals in space group P21212 with cell dimensions a = 93.8, b = 74.4, c = 36.5 Å and two molecules in the asymmetric unit (ASU) (Fig. 1A, left panel). The Mathews coefficient Vm is 2.67 Å3/Da. The NMR solution structure of wild type CV-N (PDB accession code 2EZM) (
      • Bewley C.A.
      • Gustafson K.R.
      • Boyd M.R.
      • Covell D.G.
      • Bax A.
      • Clore G.M.
      • Gronenborn A.M.
      Solution structure of cyanovirin-N, a potent HIV-inactivating protein.
      ) was used as the search model. All pertinent crystallographic statistics are provided in Table 1. Two independent monomers in close proximity were selected as the ASU and are shown in Fig. 1A. Each independent monomer in the ASU is also related to a copy of the other by pseudotranslational non-crystallographic symmetry. 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, non-domain-swapped CV-N variants (
      • Matei E.
      • Furey W.
      • Gronenborn A.M.
      Solution and crystal structures of a sugar binding site mutant of cyanovirin-N: no evidence of domain swapping.
      ,
      • Fromme R.
      • Katiliene Z.
      • Giomarelli B.
      • Bogani F.
      • Mc Mahon J.
      • Mori T.
      • Fromme P.
      • Ghirlanda G.
      A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity.
      ), in contrast to the domain-swapped wild type CV-N structures (
      • Botos I.
      • O'Keefe B.R.
      • Shenoy S.R.
      • Cartner L.K.
      • Ratner D.M.
      • Seeberger P.H.
      • Boyd M.R.
      • Wlodawer A.
      Structures of the complexes of a potent anti-HIV protein cyanovirin-N and high mannose oligosaccharides.
      ,
      • Yang F.
      • Bewley C.A.
      • Louis J.M.
      • Gustafson K.R.
      • Boyd M.R.
      • Gronenborn A.M.
      • Clore G.M.
      • Wlodawer A.
      Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping.
      ,
      • Barrientos L.G.
      • Louis J.M.
      • Botos I.
      • Mori T.
      • Han Z.
      • O'Keefe B.R.
      • Boyd M.R.
      • Wlodawer A.
      • Gronenborn A.M.
      The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of x-ray and NMR structures.
      ). We ascertained that the x-ray data were incompatible with a domain-swapped dimer structure by omitting the hinge-loop region (Trp49–Asn53) in the model employed for molecular replacement. After the first refinement step, the electron density map clearly showed strong density for the omitted region, connecting the two separate starting segments in a contiguous polypeptide chain (Fig. 1A, middle panel).
      Figure thumbnail gr1
      FIGURE 1Crystal structure of Cyt-CVNH. A, left panel, ribbon representation of the Cyt-CVNH crystal structure, illustrating the relative orientation of the two monomers (m1 and m2) in the asymmetric unit. The long loop (Leu68–Thr72) of Cyt-CVNH is colored in magenta. Center panel, view of the σA-weighted (2mFoDFc) electron density map for the loop region Trp50–Asn54, contoured at 0.5 σ. Right panel, superposition of the crystal structures of Cyt-CVNH (violet) and the domain-swapped CV-N dimer (orange; PDB accession code 3EZM), illustrating the relative orientation of the second monomer; in CV-N this is the pseudo-monomer of the dimer, and in Cyt-CVNH it is the second monomer in the asymmetric unit (gray). Residues Gln50–Thr57 constitute the hinge region (green) in the domain-swapped CV-N structure, whereas the equivalent region (Gly51–Thr58) in Cyt-CVNH adopts a loop conformation (cyan). B, left panel, surface representation of the Cyt-CVNH structure (two monomers in the asymmetric unit), illustrating the loop-mediated protein-protein contacts between adjacent monomers in the crystallographic dimer. Center panel, details of the perpendicular arrangement between the Phe69 side chain in the long Leu68–Thr72 loop (monomer 1, m1) and the Trp50 aromatic ring (monomer 2, m2). Right panel, details of the protein-protein interface between the two Cyt-CVNH monomers in the asymmetric unit. The closest interface contacts (<5 Å), including two H-bonds, are marked. C, alignment of Cyt-CVNH and CV-N amino acid sequences. The insert in Cyt-CVN-H is colored magenta, and the hinge (CV-N)-loop (Cyt-CVNH) sequences are shown in green and cyan, respectively.
      TABLE 1Data collection and refinement statistics for the x-ray structure
      Cyt-CVNH
      One crystal was used for data collection and structure determination.
      Data collection
      Space groupP21212
      Cell dimensions
      a, b, c (Å)93.8, 74.4, 36.5
      α, α, γ (°)90, 90, 90
      Resolution (Å)34.58–1.6
      ASU content (molecules)2
      Rmerge0.11(0.83)
      The values in parentheses are for the highest resolution shell.
      II23.6 (1.7)
      Completeness (%)99.8 (95.6)
      Redundancy13.1 (4.6)
      CC 1/20.98 (0.91)
      Refinement
      Resolution (Å)34.58–1.6
      No. reflections34,474
      Rwork/Rfree20.2/23.3
      No. atoms1,912
      Protein1,677
      Diethylene glycol18
      Water217
      Average B value (Å2)20.5
      Protein18.0
      Diethylene glycol34.1
      Water38.7
      Root mean square deviations
      Bond lengths (Å)0.006
      Bond angles (°)1.090
      Ramachandran statistics
      Residues in favorable regions (%)98.1
      Residues in disallowed regions (%)0
      a One crystal was used for data collection and structure determination.
      b The values in parentheses are for the highest resolution shell.
      Within the asymmetric unit, the monomers are oriented with respect to each other by an angle of ∼104° between the long axes of the two domains (AB′, A′B), using the Sγ atoms of the cysteines in the two disulfide bonds (Cys8/Cys59′;Cys59/Cys8′) to define the axes. This spatial arrangement is opposite in orientation to what was previously observed between the two halves of the domain-swapped CV-N structures (−101°) in the trigonal crystal (P3221; PDB accession code 3EZM; Fig. 1A, right panel).
      A surface view of the two molecules in the ASU is provided in Fig. 1B (left panel). The monomer-monomer interface involves a different region, with Phe69 of one monomeric unit engaged in a crystal contact with Trp50 of the adjacent monomeric unit (Fig. 1B, middle panel). The side chain of Asp45 in monomer 1 hydrogen bonds to the side chain of Lys6 in monomer 2, and similarly, the backbone carbonyl oxygen of Thr47 in monomer 1 hydrogen bonds with the side chain amide group of Gln3 in monomer 2 (Fig. 1B, right panel).

      Sugar Binding

      The carbohydrate binding sites of Cyt-CVNH were mapped by monitoring chemical shift changes in the 1H-15N HSQC spectrum of uniformly 15N-labeled protein as a function of sugar addition. Titration experiments were carried out with Man-2, Man-3, and Man-9. Interestingly, binding is in slow exchange on the chemical shift scale at 298 K, which is different from what was observed with wild type CV-N and Man2/Man3, where binding was in the fast exchange regime. Spectra of Cyt-CVNH in the absence and presence of Man-2, Man-3, and Man-9, respectively, are provided in Fig. 2. Mapping of the affected amide resonances clearly revealed that for all three sugars two binding sites exist: one on domain A and one on domain B.
      Figure thumbnail gr2
      FIGURE 2Carbohydrate binding by Cyt-CVNH. A, superposition of the 1H-15N HSQC spectra of free Cyt-CVNH (150 μm; black) and in the presence of 15-fold molar excess of Manα(1–2)Man (cyan). B, free Cyt-CVNH (50 μm; black) and in the presence of 6-fold molar excess Manα(1–2)Manα(1–2)Man (magenta). C, free Cyt-CVNH (10 μm; black) and in the presence of 2-fold molar excess Man9GlcNAc2 (magenta). Resonances of residues that undergo chemical shift changes upon carbohydrate binding are labeled in A–C. For Manα(1–2)Man, Manα(1–2)Manα(1–2)Man, and Man9GlcNAc2 binding to Cyt-CVNH the sugar-bound and -free Cyt-CVNH resonances are in slow exchange. In the right panels of A and B, the binding isotherms (bound state signal intensity versus ligand/protein molar ratio) for each domain are shown. In the right panel of C, the chemical structure of Man9GlcNAc2 is shown. The D1 arm (magenta) contains the Manα(1–2)Manα(1–2)Man trimannoside, whereas the D3 arm (cyan) contains the Manα(1–2)Man dimannoside.
      A superposition of the spectra of Cyt-CVNH in the absence (black contours) and presence (cyan contours) of 15 molar equivalents of Man-2 are provided in Fig. 2A. Likewise, superposition of free Cyt-CVNH (black contours) and protein in the presence of 6 molar equivalents of Man-3 or 2 molar equivalents of Man-9 (magenta) are provided in Fig. 2 (B and C, respectively). The extracted binding isotherms derived from the intensity changes of sugar-bound amide resonances of residues from domain A and B are depicted in the right panels. The dissociation constants for Man-2 binding to the sites on domain A and B are 53.2 ± 8.7 and 48.4 ± 9.5 μm, respectively. The equivalent Kd values for Man-3 are 7.5 ± 1.2 and 9.4 ± 0.8 μm, respectively.
      In the past, attempts to structurally monitor Man-9 binding to CV-N by NMR were hampered by extreme line broadening and ultimately disappearance of resonances in the 1H-15N HSQC spectra, accompanied by precipitation of the sugar· protein caused by multisite/multivalent cross-linking (
      • Shenoy S.R.
      • Barrientos L.G.
      • Ratner D.M.
      • O'Keefe B.R.
      • Seeberger P.H.
      • Gronenborn A.M.
      • Boyd M.R.
      Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.
      ,
      • Matei E.
      • Zheng A.
      • Furey W.
      • Rose J.
      • Aiken C.
      • Gronenborn A.M.
      Anti-HIV activity of defective cyanovirin-N mutants is restored by dimerization.
      ,
      • Barrientos L.G.
      • Matei E.
      • Lasala F.
      • Delgado R.
      • Gronenborn A.M.
      Dissecting carbohydrate-cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
      ). In contrast to the findings with CV-N, no aggregation or precipitation was observed for Man-9 binding to Cyt-CVNH. Therefore, it was possible to identify those amide resonances that were affected by Man-9 binding. Again, two binding sites are present, and Kd values of ∼500 nm were obtained for domain A and B. Thus, domain A and domain B of Cyt-CVNH possess essentially the same affinities for Man-9. The same holds for Man-2 or Man-3.
      Because the D1 and D3 arms of Man-9 contain α1→2-linked mannoses, a single molecule of Man-9 can interact with more than one lectin molecule that recognizes Manα(1–2)Manα units. The glycan binding site of CV-N in domain A exhibits a slight preference for Man-3, whereas domain B preferentially binds Man-2, resulting in cross-linking when interacting with Man-9 (
      • Shenoy S.R.
      • Barrientos L.G.
      • Ratner D.M.
      • O'Keefe B.R.
      • Seeberger P.H.
      • Gronenborn A.M.
      • Boyd M.R.
      Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.
      ,
      • Matei E.
      • Zheng A.
      • Furey W.
      • Rose J.
      • Aiken C.
      • Gronenborn A.M.
      Anti-HIV activity of defective cyanovirin-N mutants is restored by dimerization.
      ,
      • Barrientos L.G.
      • Matei E.
      • Lasala F.
      • Delgado R.
      • Gronenborn A.M.
      Dissecting carbohydrate-cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
      ). Thus, Man-9 interacts with CV-N with the D1 arm, engaging domain A, and the D3 arm, binding to domain B. For Cyt-CVNH, we noticed in the titration that Man-9 binding elicited changes in the 1H-15N HSQC spectrum very similar to those of Man-3 and somewhat different from Man-2 (Fig. 3). For instance, at a 0.5:1 Man-2:Cyt-CVNH molar ratio the Thr58 resonance (domain B) is shifted, whereas the Thr7 resonance (domain A) is not affected (Fig. 3A). This is different for Man-3 and Man-9 binding, where the Thr7 resonance (domain A) is affected, but not the Thr58 resonance (Fig. 3, B and C). Furthermore, at saturation, i.e. when both binding sites on the protein are sugar-bound, the pattern observed for the Man-3 and Man-9 shifted resonances is very similar for the Thr7 resonance (Fig. 3, B and C, right panels) and distinctly different from the one observed upon Man-2 binding (Fig. 3A, right panel). This similarity between the Man-3 and Man-9 effects is observed throughout the entire titration, for resonances belonging to both domains A and B (supplemental Figs. S1 and S2). For instance, at a 1:1 sugar-protein molar ratio, the Thr86 resonance (domain B) clearly shows a smaller chemical shift difference between free and sugar-bound protein for Man-2 binding, compared with Man-3 or Man-9. In addition, Man-3 exhibits ∼6-fold higher affinity compared with Man-2, for both binding sites. These suggest that Man-3 and Man-9 interact in similar fashion with domain A and domain B of Cyt-CVNH. At a 1:1 molar ratio of sugar:protein, amide resonances of the majority of residues in the binding site of domain A, including Gly2, Gln3, Thr7, Thr29, Leu30, Gln23, Asp102, Gly103, and Thr104 undergo chemical shift changes only upon Man-3 and Man-9 binding, whereas two residues, Phe4 and Lys24, exhibit perturbed resonances for all three sugars (Man-2, Man-3, and Man-9). Similarly, resonances of residues in domain B, such as Gly46, Arg81, Asp83, and Thr86 undergo chemical shift changes upon Man-3 and Man-9 binding at 1:1 molar ratio of sugar:protein, whereas Gly42, Leu44, Gly45, Leu48, Trp50, and His53 are affected by Man-2, Man-3, and Man-9 binding, with Asp52, Asp54, and Phe55 affected only by Man-2 binding.
      Figure thumbnail gr3
      FIGURE 3Oligomannose-9 binding to Cyt-CVNH. A–C, selected region of the superimposed 1H-15N HSQC spectra, showing the resonances of Thr7 (domain A) and Thr58 (domain B) in the free protein (black) and in the presence of 0.5, 1, and 2 equivalents of Man2 (A, cyan), Man3 (B, magenta), and Man9 (C, magenta). D, schematic illustration depicting Man-9 binding to Cyt-CVNH via the D1 arm. The protein is represented in orange with the binding sites on domain A (site 1) and domain B (site 2) are colored green and pink, respectively. The individual sugar units of the oligosaccharide are color-coded according to their linkage pattern.
      This is illustrated diagrammatically in the scheme of Man-9 interacting with domains A and B of Cyt-CVNH (Fig. 3D). In this scheme, Man-9 interacts with domains A and B of Cyt-CVNH through its D1 arm only (Fig. 3D and supplemental Figs. S3 and S4A), and it is likely that this type of recognition, which is different from what is observed for CV-N (supplemental Fig. S4B), prevents cross-linking of Cyt-CVNH by Man-9.
      To corroborate that indeed the Man9·Cyt-CVNH complex comprises one protein and two sugars, we carried out NMR relaxation measurements. Heteronuclear T2 values for Cyt-CVNH and the Man-9·Cyt-CVNH complex were determined, yielding average T2 values of 106 ± 4.5 and 71 ± 3.7 ms, respectively. These values are consistent with the expected mass of ∼12 kDa for the protein and with the ∼16 kDa mass matching the 2:1 Man-9·Cyt-CVNH complex (
      • Bloembergen N.
      • Purcell E.M.
      • Pound R.V.
      Relaxation effects in nuclear magnetic resonance absorption.
      ).

      HIV Assays

      To determine the antiviral potency of the new lectin, Cyt-CVNH was assessed in parallel with monomeric CV-NP51G, a stabilized version of wild type CV-N (
      • Barrientos L.G.
      • Louis J.M.
      • Botos I.
      • Mori T.
      • Han Z.
      • O'Keefe B.R.
      • Boyd M.R.
      • Wlodawer A.
      • Gronenborn A.M.
      The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of x-ray and NMR structures.
      ), in a single-cycle luciferase reporter assay. This assay relies on completion of the early steps in infection, including entry, reverse transcription, integration, and expression of the viral Tat protein.
      Normalized inhibition curves for concentrations up to 100 nm are provided in Fig. 4. An IC50 value of ∼0.175 ± 0.01 nm was extracted for Cyt-CVNH, compared with an IC50 value of ∼0.7 ± 0.02 nm for CV-NP51G. Thus, Cyt-CVNH exhibits ∼4-fold higher activity than CV-NP51G.
      Figure thumbnail gr4
      FIGURE 4Anti-HIV activity. Single-cycle HIV-1 infectivity assays carried out with Cyt-CVNH (orange-filled circles) and CV-NP51G (black open circles), using a luciferase reporter cell line. The symbols are experimental data points with the best fit to the data shown as continuous lines.

      Discussion

      The envelope glycoprotein gp120 of HIV-1 mediates host cell entry, which is initiated by engaging the host cell CD4 receptor. This causes a conformational change in gp120, resulting in co-receptor (CCR5 or CXCR4) binding and ultimately fusion of the viral and cellular membranes (
      • Kuritzkes D.R.
      HIV-1 entry inhibitors: an overview.
      ,
      • Briz V.
      • Poveda E.
      • Soriano V.
      HIV entry inhibitors: mechanisms of action and resistance pathways.
      ,
      • Castagna A.
      • Biswas P.
      • Beretta A.
      • Lazzarin A.
      The appealing story of HIV entry inhibitors: from discovery of biological mechanisms to drug development.
      ). Gp120 is highly glycosylated, containing a large number of high mannose sugars (
      • Lasky L.A.
      • Groopman J.E.
      • Fennie C.W.
      • Benz P.M.
      • Capon D.J.
      • Dowbenko D.J.
      • Nakamura G.R.
      • Nunes W.M.
      • Renz M.E.
      • Berman P.W.
      Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein.
      ,
      • Leonard C.K.
      • Spellman M.W.
      • Riddle L.
      • Harris R.J.
      • Thomas J.N.
      • Gregory T.J.
      Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.
      ,
      • Pritchard L.K.
      • Vasiljevic S.
      • Ozorowski G.
      • Seabright G.E.
      • Cupo A.
      • Ringe R.
      • Kim H.J.
      • Sanders R.W.
      • Doores K.J.
      • Burton D.R.
      • Wilson I.A.
      • Ward A.B.
      • Moore J.P.
      • Crispin M.
      Structural constraints determine the glycosylation of HIV-1 envelope trimers.
      ,
      • Ward A.B.
      • Wilson I.A.
      Insights into the trimeric HIV-1 envelope glycoprotein structure.
      ), and the glycosylation sites are well conserved (
      • Kwong P.D.
      • Wyatt R.
      • Robinson J.
      • Sweet R.W.
      • Sodroski J.
      • Hendrickson W.A.
      Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody.
      ). A number of broadly neutralizing antibodies (Abs) target glycans on gp120 (
      • Walker L.M.
      • Huber M.
      • Doores K.J.
      • Falkowska E.
      • Pejchal R.
      • Julien J.P.
      • Wang S.K.
      • Ramos A.
      • Chan-Hui P.Y.
      • Moyle M.
      • Mitcham J.L.
      • Hammond P.W.
      • Olsen O.A.
      • Phung P.
      • Fling S.
      • et al.
      Broad neutralization coverage of HIV by multiple highly potent antibodies.
      ,
      • Scharf L.
      • Scheid J.F.
      • Lee J.H.
      • West Jr., A.P.
      • Chen C.
      • Gao H.
      • Gnanapragasam P.N.
      • Mares R.
      • Seaman M.S.
      • Ward A.B.
      • Nussenzweig M.C.
      • Bjorkman P.J.
      Antibody 8ANC195 reveals a site of broad vulnerability on the HIV-1 envelope spike.
      ,
      • Huang J.
      • Kang B.H.
      • Pancera M.
      • Lee J.H.
      • Tong T.
      • Feng Y.
      • Imamichi H.
      • Georgiev I.S.
      • Chuang G.Y.
      • Druz A.
      • Doria-Rose N.A.
      • Laub L.
      • Sliepen K.
      • van Gils M.J.
      • de la Peña A.T.
      • et al.
      Broad and potent HIV-1 neutralization by a human antibody that binds the gp41–gp120 interface.
      ) and potentially could be used to combat HIV-1 infection. 2G12 was one of the first such antibodies described (
      • Scanlan C.N.
      • Pantophlet R.
      • Wormald M.R.
      • Ollmann Saphire E.
      • Stanfield R.
      • Wilson I.A.
      • Katinger H.
      • Dwek R.A.
      • Rudd P.M.
      • Burton D.R.
      The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of a1–2 mannose residues on the outer face of gp120.
      ,
      • Sanders R.W.
      • Venturi M.
      • Schiffner L.
      • Kalyanaraman R.
      • Katinger H.
      • Lloyd K.O.
      • Kwong P.D.
      • Moore J.P.
      The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120.
      ,
      • Calarese D.A.
      • Scanlan C.N.
      • Zwick M.B.
      • Deechongkit S.
      • Mimura Y.
      • Kunert R.
      • Zhu P.
      • Wormald M.R.
      • Stanfield R.L.
      • Roux K.H.
      • Kelly J.W.
      • Rudd P.M.
      • Dwek R.A.
      • Katinger H.
      • Burton D.R.
      • et al.
      Antibody domain exchange is an immunological solution to carbohydrate cluster recognition.
      ), followed more recently by a collection of Abs, including PGT121, PGT122, PGT128, and PGT135, that recognize dual protein-glycan epitopes, especially involving the sugar on Asn332 (
      • Walker L.M.
      • Huber M.
      • Doores K.J.
      • Falkowska E.
      • Pejchal R.
      • Julien J.P.
      • Wang S.K.
      • Ramos A.
      • Chan-Hui P.Y.
      • Moyle M.
      • Mitcham J.L.
      • Hammond P.W.
      • Olsen O.A.
      • Phung P.
      • Fling S.
      • et al.
      Broad neutralization coverage of HIV by multiple highly potent antibodies.
      ,
      • Pejchal R.
      • Doores K.J.
      • Walker L.M.
      • Khayat R.
      • Huang P.S.
      • Wang S.K.
      • Stanfield R.L.
      • Julien J.P.
      • Ramos A.
      • Crispin M.
      • Depetris R.
      • Katpally U.
      • Marozsan A.
      • Cupo A.
      • Maloveste S.
      • et al.
      A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.
      ,
      • Mouquet H.
      • Scharf L.
      • Euler Z.
      • Liu Y.
      • Eden C.
      • Scheid J.F.
      • Halper-Stromberg A.
      • Gnanapragasam P.N.
      • Spencer D.I.
      • Seaman M.S.
      • Schuitemaker H.
      • Feizi T.
      • Nussenzweig M.C.
      • Bjorkman P.J.
      Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies.
      ,
      • Julien J.-P.
      • Julien J.P.
      • Sok D.
      • Khayat R.
      • Lee J.H.
      • Doores K.J.
      • Walker L.M.
      • Ramos A.
      • Diwanji D.C.
      • Pejchal R.
      • Cupo A.
      • Katpally U.
      • Depetris R.S.
      • Stanfield R.L.
      • McBride R.
      • Marozsan A.J.
      • et al.
      Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.
      ,
      • Sok D.
      • Doores K.J.
      • Briney B.
      • Le K.M.
      • Saye-Francisco K.L.
      • Ramos A.
      • Kulp D.W.
      • Julien J.P.
      • Menis S.
      • Wickramasinghe L.
      • Seaman M.S.
      • Schief W.R.
      • Wilson I.A.
      • Poignard P.
      • Burton D.R.
      Promiscuous glycan site recognition by antibodies to the highmannose patch of gp120 broadens neutralization of HIV.
      ). Crystal structures of glycosylated simian immunodeficiency virus gp120 (Ref.
      • Chen B.
      • Vogan E.M.
      • Gong H.
      • Skehel J.J.
      • Wiley D.C.
      • Harrison S.C.
      Determining the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein.
      ; PDB accession code 2BF1), as well as a HIV-1 trimer, complexed with the PGT122 antibody (Ref.
      • Julien J.P.
      • Cupo A.
      • Sok D.
      • Stanfield R.L.
      • Lyumkis D.
      • Deller M.C.
      • Klasse P.J.
      • Burton D.R.
      • Sanders R.W.
      • Moore J.P.
      • Ward A.B.
      • Wilson I.A.
      Crystal structure of a soluble cleaved HIV-1 envelope trimer.
      ; PDB accession code 4CNO) are available. Importantly, the Asn332 glycan is 73% conserved among HIV isolates. The crystal structure of the gp120 trimer clearly delineates the Asn332 (Man8/9GlcNAc2) glycan as a key element in PGT122 recognition (
      • Pejchal R.
      • Doores K.J.
      • Walker L.M.
      • Khayat R.
      • Huang P.S.
      • Wang S.K.
      • Stanfield R.L.
      • Julien J.P.
      • Ramos A.
      • Crispin M.
      • Depetris R.
      • Katpally U.
      • Marozsan A.
      • Cupo A.
      • Maloveste S.
      • et al.
      A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.
      ,
      • Julien J.P.
      • Cupo A.
      • Sok D.
      • Stanfield R.L.
      • Lyumkis D.
      • Deller M.C.
      • Klasse P.J.
      • Burton D.R.
      • Sanders R.W.
      • Moore J.P.
      • Ward A.B.
      • Wilson I.A.
      Crystal structure of a soluble cleaved HIV-1 envelope trimer.
      ).
      Akin to mannose-targeting Abs, lectins also interact with sugars, and several mannose-targeting lectins have been shown to possess virucidal properties against HIV. CV-N, discovered as one of the first HIV-1-inactivating lectins, is active against HIV-1 and -2, simian immunodeficiency virus, and other enveloped viruses like Ebola and Influenza (
      • Bewley C.A.
      • Gustafson K.R.
      • Boyd M.R.
      • Covell D.G.
      • Bax A.
      • Clore G.M.
      • Gronenborn A.M.
      Solution structure of cyanovirin-N, a potent HIV-inactivating protein.
      ) at nanomolar concentrations (
      • Barrientos L.G.
      • Gronenborn A.M.
      The highly specific carbohydrate-binding protein cyanovirin-N: structure, anti-HIV/Ebola activity and possibilities for therapy.
      ). Previous biochemical and biophysical studies revealed that two binding sites for Manα(1–2)Manα-containing sugars are located on CV-N: one each on domains A and B. It was shown that CV-N recognizes the terminal Manα(1–2)Manα units of both the D1 and the D3 arms of Man-8 and Man-9 as the primary target (
      • Bolmstedt A.J.
      • O'Keefe B.R.
      • Shenoy S.R.
      • McMahon J.B.
      • Boyd M.R.
      Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner.
      ,
      • Shenoy S.R.
      • O'Keefe B.R.
      • Bolmstedt A.J.
      • Cartner L.K.
      • Boyd M.R.
      Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins.
      ,
      • Bewley C.A.
      • Otero-Quintero S.
      The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.
      ,
      • Botos I.
      • O'Keefe B.R.
      • Shenoy S.R.
      • Cartner L.K.
      • Ratner D.M.
      • Seeberger P.H.
      • Boyd M.R.
      • Wlodawer A.
      Structures of the complexes of a potent anti-HIV protein cyanovirin-N and high mannose oligosaccharides.
      ,
      • Sandström C.
      • Berteau O.
      • Gemma E.
      • Oscarson S.
      • Kenne L.
      • Gronenborn A.M.
      Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR.
      ). Likewise, members of the CVNH family from Tuber borchii (TbCVNH), Ceratopteris richardii (CrCVNH), Neurospora crassa (NcCVNH), and Gibberella zeae (GzCVNH) also recognize Manα(1–2)Man disaccharides, but with lower affinity than CV-N (
      • Koharudin L.M.
      • Viscomi A.R.
      • Jee J.G.
      • Ottonello S.
      • Gronenborn A.M.
      The evolutionary conserved family of cyanovirin-N homologs (CVNHs): Structures and carbohydrate specificity.
      ,
      • Matei E.
      • Louis J.M.
      • Jee J.
      • Gronenborn A.M.
      NMR solution structure of a cyanovirin homolog from wheat head blight fungus.
      ). Each of these proteins exhibits carbohydrate binding sites that are different in number and location. CrCVNH possesses two sites; TbCVNH possesses a single binding site on domain A, whereas GzCVNH and NcCVNH have one site only on domain B. With the exception of GzCVNH, carbohydrate binding specificities are distinct as well, and no potent HIV inactivation was observed with any of these proteins (
      • Koharudin L.M.
      • Viscomi A.R.
      • Jee J.G.
      • Ottonello S.
      • Gronenborn A.M.
      The evolutionary conserved family of cyanovirin-N homologs (CVNHs): Structures and carbohydrate specificity.
      ,
      • Matei E.
      • Louis J.M.
      • Jee J.
      • Gronenborn A.M.
      NMR solution structure of a cyanovirin homolog from wheat head blight fungus.
      ).
      Although in principle very powerful for delineating binding sites on proteins, using 1H-15N HSQC spectroscopy for following Man-9 binding to CV-N, even at low concentration, was impossible because precipitation of the sugar·protein complex occurred, caused by multisite/multivalent cross-linking (
      • Shenoy S.R.
      • Barrientos L.G.
      • Ratner D.M.
      • O'Keefe B.R.
      • Seeberger P.H.
      • Gronenborn A.M.
      • Boyd M.R.
      Multisite and multivalent binding between cyanovirin-N and branched oligomannosides: calorimetric and NMR characterization.
      ,
      • Matei E.
      • Zheng A.
      • Furey W.
      • Rose J.
      • Aiken C.
      • Gronenborn A.M.
      Anti-HIV activity of defective cyanovirin-N mutants is restored by dimerization.
      ,
      • Barrientos L.G.
      • Matei E.
      • Lasala F.
      • Delgado R.
      • Gronenborn A.M.
      Dissecting carbohydrate-cyanovirin-N binding by structure-guided mutagenesis: functional implications for viral entry inhibition.
      ). Here, by contrast, for Cyt-CVNH, no aggregation or precipitation was seen for the Man9·Cyt-CVNH complex. In addition, based on the patterns observed throughout the titrations with Man-2, Man-3, and Man-9, it is evident that Man-9 binds both sites on Cyt-CVNH through its D1 arm only (Fig. 3D). Thus, unlike for CV-N, where 1:1 binding between Man-9 and protein is causing precipitation, for Cyt-CVNH a 2:1 sugar·protein complex is formed, without any cross-linked higher molecular species noted.
      Based on all available data, we developed an interaction model for CVNH lectins and glycosylated gp120, assuming that the interaction involves the glycan on Asn332. As the starting model, we used the crystal structure of the HIV-1 Env trimer in complex with the antibody PGT122 (blue; PDB accession code 4NCO; the antibody coordinates are omitted from one of the gp120 monomeric units). Onto the trimer, we superimposed the monomeric gp120 core structure (orange) in complex with CD4 (magenta; PDB accession code 3JWD) (Fig. 5, top panel). As can be appreciated, the Env trimer structure contains a large number of sugar molecules. In addition to Asn332, which bears Man8/Man9, the PGT122 Ab interacts with the base of the V1 and V3 loops on the protein and three more glycans on Asn301, Asn156/Asn173, and Asn137 (
      • Julien J.P.
      • Cupo A.
      • Sok D.
      • Stanfield R.L.
      • Lyumkis D.
      • Deller M.C.
      • Klasse P.J.
      • Burton D.R.
      • Sanders R.W.
      • Moore J.P.
      • Ward A.B.
      • Wilson I.A.
      Crystal structure of a soluble cleaved HIV-1 envelope trimer.
      ).
      Figure thumbnail gr5
      FIGURE 5Model for the interaction between CVNH lectins and glycosylated gp120. Top panel, the crystal structure of the HIV-1 Env gp120 trimer (blue) in complex with the antibody PGT122 (gray; PDB accession code 4NCO) was superimposed on the crystal structure of the monomeric gp120 core (orange) bound to CD4 (magenta; PDB accession code 3JWD). The gp41 trimer is shown in yellow. Gp120 glycosylation sites are labeled on one monomer, and the sugars are shown in blue stick representation. The x-ray structure of Cyt-CVNH bound to one monomer is shown in red, with the sugars on Asn332 and Asn156 bound to domain A and domain B, respectively. Bottom panel, expanded view of the modeled interaction between the Man-8/Man-9 sugar on Asn332 and Asn156 and Cyt-CVNH. The epitope used by the lectin is located on the opposite side of the CD4 binding site, similar to where PGT122 is bound.
      The distance between the two glycan binding sites on CVNH lectins is ∼40 Å. If the D1 arm of Man-8/Man-9 on Asn332 is binding one of these two sites, a possible second sugar on Asn156 could interact with the second site. First, we placed the Cyt-CVNH x-ray structure onto the Asn332 glycosylation site. This allows Man8/Man-9 to interact with domain A. Next we rotated the Cyt-CVNH model around the Asn332 sugar, to find another possible sugar on gp120 that could interact with domain B; this was the sugar on Asn156. Using the sugars on Asn156 and Asn332 (Fig. 5, bottom panel), the CVNH lectin is contacting approximately the same region as the PGT122 antibody.
      It is well established that interaction of CD4 with the HIV envelope causes conformational changes in the trimer, leading to a more open conformation that exposes the co-receptor binding site (
      • Liu J.
      • Bartesaghi A.
      • Borgnia M.J.
      • Sapiro G.
      • Subramaniam S.
      Molecular architecture of native HIV-1 gp120 trimers.
      ). Although steric occlusion of the CD4 binding site by PGT121 binding to gp120 does not seem to be the mechanism for competitive inhibition, allosteric effects that interfere with the CD4 induced conformational changes may play a role (
      • Mouquet H.
      • Scharf L.
      • Euler Z.
      • Liu Y.
      • Eden C.
      • Scheid J.F.
      • Halper-Stromberg A.
      • Gnanapragasam P.N.
      • Spencer D.I.
      • Seaman M.S.
      • Schuitemaker H.
      • Feizi T.
      • Nussenzweig M.C.
      • Bjorkman P.J.
      Complex-type N-glycan recognition by potent broadly neutralizing HIV antibodies.
      ). Such mechanisms may also apply to glycan-binding lectins, although other mechanisms cannot be ruled out at present, and further studies are necessary to determine whether CVNH lectins are capable of preventing CD4 binding through an allosteric mechanism. Alternatively, they may exert their antiviral activity by inducing post-binding conformational effects that prevent CD4-bound gp120 from interacting with CCR5 or CXCR4 co-receptors.

      Experimental Procedures

      Protein Expression and Purification

      The protein was expressed from a synthetic gene, using pET26b(+) (Novagen; Madison, WI) and Escherichia coli BL21(DE3) as expression vector and host strain, respectively. The cells were initially grown at 37 °C, induced with 1 mm isopropyl β-d-thiogalactopyranoside at 16 °C, and grown for ∼12 h at 16 °C for protein expression. Uniform 15N and 13C labeling of Cyt-CVNH was carried out by growth in modified minimal medium, using 15NH4Cl and 13C6-glucose as the sole nitrogen and carbon sources, respectively. The cells were harvested by centrifugation (4600 × g for 15 min at 4 °C), resuspended in 20 mm potassium phosphate buffer, pH 6.0, and lysed using a mircrofluidizer (MicroFluidics M-110Y, Hyland Scientific). Cell debris was removed by ultracentrifugation (120,000 × g), and the supernatant was fractionated by gel filtration on a Superdex 75 (HiLoad 2.6 × 60 cm; Amersham Biosciences) column, equilibrated in 20 mm sodium phosphate buffer, pH 6.0. Protein fractions containing Cyt-CVNH were collected and concentrated up to 10 mg/ml using Centriprep devices (Millipore). Protein purity was estimated >99% by SDS-PAGE and mass spectrometry.

      Crystallization and X-ray Data Collection

      Cyt-CVNH protein was crystallized by sitting drop vapor diffusion from a 1.0 mm protein solution in 20 mm sodium phosphate buffer, 0.01% NaN3, pH 6.0. The best crystals were obtained at room temperature in 0.2 m magnesium chloride hexahydrate, 0.1 m Bis-Tris, pH 5.5, with 25% (w/v) PEG 3350 and 15% ethylene glycol as precipitants. Crystal growth took ∼30 days, yielding crystals with dimensions of 0.20 × 0.30 × 0.70 mm. X-ray diffraction data were collected from a single flash-cooled crystal (−180 °C) at the Southeast Regional Collaborative Access Team facility sector 22-ID beam line of the Advance Photon Source (Argonne National Laboratory, Chicago, IL). 451,609 total observations were reduced to yield 34,474 unique reflections (98% complete), with a 13.1 redundancy, to 1.6 Å resolution, with an internal R factor (based on intensities) of 0.11. The data were processed and scaled with the HKL2000 package (
      • Otwinowski Z.
      • Minor W.
      Processing of x-ray diffraction data collected in oscillation mode.
      ).

      Crystal Structure Determination and Refinement

      The crystal structure of Cyt-CVNH was solved by molecular replacement in Phenix (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.-W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ), using the monomeric NMR structure of wild type CV-N (PDB accession code 2EZM) (
      • Bewley C.A.
      • Gustafson K.R.
      • Boyd M.R.
      • Covell D.G.
      • Bax A.
      • Clore G.M.
      • Gronenborn A.M.
      Solution structure of cyanovirin-N, a potent HIV-inactivating protein.
      ) as the search model. The initial model included two independent segments of the chain, comprising residues Leu1–Lys48 and Phe54–Glu101, with the hinge-loop region (Trp49–Asn53) omitted. Iterative rigid body and simulated annealing refinement in Phenix was alternated with model building, including the hinge-loop region, in Coot (
      • Emsley P.
      • Cowtan K.
      Coot: model-building tools for molecular graphics.
      ). The final stages of refinement included periodic examinations of σA-weighted electron density (2mFoDFc) and difference electron density (mFoDFc) maps, as well as the introduction of water and several cryoprotectant molecules. Analysis of the final structural model was performed using PROCHECK (
      • Laskowski R.A.
      • Rullmannn J.A.
      • MacArthur M.W.
      • Kaptein R.
      • Thornton J.M.
      AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR.
      ). Approximately 98% of all residues reside in the favored region of the Ramachandran plot (
      • Ramachandran G.N.
      • Sasisekharan V.
      Conformation of polypeptides and proteins.
      ) with no residues in the disallowed regions. The final model was also validated by MolProbity (
      • Davis I.W.
      • Murray L.W.
      • Richardson J.S.
      • Richardson D.C.
      MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes.
      ), with an overall clash score of 0.31 and percentile of 100%. Atomic coordinates and structure factors have been deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession code 5K79. All structural figures were generated with PyMOL (
      • DeLano W.L.
      ).

      NMR Spectroscopy

      NMR spectra were recorded at 298 K on Bruker 600 MHz and 800 MHz AVANCE spectrometers, equipped with 5 mm, triple resonance, three-axis gradient probes, or z axis gradient cryoprobes. For three-dimensional NMR experiments, the sample contained 0.5 mm protein in 20 mm sodium phosphate buffer, pH 6.0. For chemical shift assignments, a series of heteronuclear, multidimensional experiments, routinely used in our laboratory, were recorded (
      • Clore G.M.
      • Gronenborn A.M.
      Determination of three-dimensional structures of proteins in solution by nuclear magnetic resonance spectroscopy.
      ,
      • Bax A.
      • Grzesiek S.
      Methodological advances in protein NMR.
      ,
      • Fesik S.W.
      • Zuiderweg E.R.
      Heteronuclear three-dimensional NMR spectroscopy of isotopically labelled biological macromolecules.
      ,
      • Mori S.
      • Abeygunawardana C.
      • Johnson M.O.
      • van Zijl P.C.
      Improved sensitivity of HSQC spectra of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection scheme that avoids water saturation.
      ). Complete 1H, 15N, and 13C backbone resonance assignments were obtained using NMR data obtained from two-dimensional 1H-15N HSQC, three-dimensional HNCACB, HN(CO)CACB, HNCA, and HN(CO)CA spectra.

      Binding Studies

      Binding of sugar to protein was assessed in titration experiments, using uniformly 15N-labeled Cyt-CVNH (150 μm) at 298 K in 20 mm sodium phosphate buffer, pH 6.0, 0.01% sodium azide, and 90% H2O/10% D2O, monitoring the chemical shift changes in 1H-15N HSQC spectra upon sugar addition. For dimannose (Man-2) binding, aliquots of a 50 mm Man-2 stock solution were added to yield sugar/protein molar ratios of: 0, 0.5, 1, 2, 3, 4, 5, 7, 10, and 15. Analogous NMR titration experiments were performed with trimannose (Man-3), using aliquots of a 10 mm stock solution to yield sugar/protein molar ratios of: 0, 0.5, 1, 2, 3, 4, 5, and 6. The protein concentration in the titrations with oligomannose-9 (Man-9) was 10 μm, and aliquots from a 500 μm stock solution were added to yield sugar/protein molar ratios of: 0, 0.5, 1, and 2.
      Free and sugar-bound protein resonances are in slow exchange on the chemical shift scale for all three sugars. The observed signal intensity of the sugar-bound protein resonances during the titration is directly proportional to the bound fraction (fb) and is given as fb = Ib/Ib max = [PL]/[P], where [P] is the total concentration of protein, [PL] is the concentration of the protein·ligand complex, Ib is the intensity of the sugar-bound protein signal at each point in the titration, and Ib max is the maximum sugar-bound protein signal intensity at the end of the titration. Binding curves were derived from the sugar-bound protein resonance intensities (Ib) versus the molar ratio (M) of sugar/protein, and apparent KD values were obtained by nonlinear best fitting of the titration curves using KaleidaGraph (Synergy Software, Reading, PA) and the following equation:
      fb=lb/lbmax=0.5*(M+1+KD[P](M+1+KD[P])24M)
      (Eq. 1)


      Anti-HIV Assay

      HIV-1 infectivity was assayed as described previously (
      • Yang R.
      • Aiken C.
      A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA.
      ). For antiviral assays, recombinant proteins were serially diluted in sterile phosphate-buffered saline, and 5 μl were added to 500 μl of prediluted infectious HIV-1 (produced by transfection of 293T cells with the R9 molecular clone and incubated for 30 min at room temperature). Aliquots of the mixture (125 μl, in duplicate) were added to cultures of TZM-bl cells (20,000 cells seeded per well the day before in a 48-well format), and after 2 days, cells were lysed and assayed for luciferase activity as previously described (
      • Shi J.
      • Zhou J.
      • Halambage U.D.
      • Shah V.B.
      • Burse M.J.
      • Wu H.
      • Blair W.S.
      • Butler S.L.
      • Aiken C.
      Compensatory substitutions in the HIV-1 capsid reduce the fitness cost associated with resistance to a capsid-targeting small-molecule inhibitor.
      ). IC50 values were determined by nonlinear best fitting of the normalized inhibition curves using KaleidaGraph (Synergy Software, Reading, PA). The results are representative of two independent experiments.

      Author Contributions

      E. M. and A. M. G. conceived and coordinated the study. R. B. performed NMR assignments and helped with NMR titrations and the preparation of figures, C. C. performed crystal optimization, and W. F. collected the diffraction data at the Advance Photon Source and was involved in x-ray data interpretation. J. S. and C. A. performed the HIV assays. E. M. and A. M. G. wrote the paper, and all authors approved its final version.

      Acknowledgments

      We thank Mike Delk for NMR technical support and Palaniappa Arjunan for help with x-ray diffraction data collection at the Advance Photon Source.

      Supplementary Material

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