structureandglycanbindingofanewcyanovirin-n …definition. the current structure of cyt-cvnh is...

Structure and Glycan Binding of a New Cyanovirin-NHomolog*□S

Received for publication, May 26, 2016, and in revised form, June 30, 2016 Published, JBC Papers in Press, July 7, 2016, DOI 10.1074/jbc.M116.740415

Elena Matei‡, Rohan Basu‡§, William Furey¶�, Jiong Shi**, Conor Calnan‡ ‡‡, Christopher Aiken**,and Angela M. Gronenborn‡1

From the ‡Department of Structural Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15260, the§Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, the ¶Department of Pharmacology& Chemical Biology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261, �Biocrystallography Laboratory,Veterans Affairs Medical Center, Pittsburgh, Pennsylvania 15240, the **Department of Pathology, Microbiology and Immunology,Vanderbilt University Medical Center, Nashville, Tennessee, 37232, and the ‡‡Department of Bioengineering, University ofPittsburgh, Pittsburgh, Pennsylvania 15261

The HIV-1 envelope glycoprotein gp120 is heavily glycosy-lated and bears numerous high mannose sugars. These sugarscan serve as targets for HIV-inactivating compounds, such asantibodies and lectins, which bind to the glycans and interferewith viral entry into the target cell. We determined the 1.6 Ax-ray structure of Cyt-CVNH, a recently identified lectin fromthe cyanobacterium Cyanothece7424, and elucidated its glycanspecificity by NMR. The Cyt-CVNH structure and glycan recog-nition profile are similar to those of other CVNH proteins, witheach domain specifically binding to Man�(1–2)Man� units onthe D1 and D3 arms of high mannose glycans. However, in con-trast 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 ofMan-9 with a member of the CVNH family by NMR. HIV assaysshowed that Cyt-CVNH is able to inhibit HIV-1 with �4-foldhigher potency than CV-NP51G, a stabilized version of wild typeCV-N. Therefore, Cyt-CVNH may qualify as a valuable lectin forpotential microbicidal use.

Despite the effective use of anti-retroviral therapies as ameans to treat HIV infection and prolong the lifespan of thoseaffected by AIDS, the number of HIV infections worldwide con-tinues to grow. Unfortunately, at present, no vaccine is availableto protect against HIV, creating the need to develop safe, effec-tive, and acceptable prevention strategies that will help halt thespread of HIV infection globally. Promising candidates forinclusion into microbicides are lectins, which are carbohy-drate-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 (1– 4).One such example is the extensively explored cyanobacteriallectin cyanovirin-N (CV-N),2 which possesses virucidal prop-erties against HIV types I and II, simian immunodeficiencyvirus, and other enveloped viruses like Ebola and influenza atnanomolar concentrations (5–7). Its anti-HIV activity is medi-ated by recognizing and interacting with high mannose glycans(Man-8, Man-9) that are present on the envelope glycoproteingp120. Indeed, HIV-1 gp120 is highly glycosylated, andN-linked glycans account for approximately half of its molecu-lar mass (8, 9). CV-N specifically binds the terminal Man�(1–2)Man� epitopes on the D1 and D3 arms of Man-8 and Man-9glycans (10 –14). The mechanism of action for mannose-bind-ing lectins is assumed to involve the inability of the lectin-bound gp120 to productively engage the host cell CD4 andCCR5 receptors, effectively preventing the necessary confor-mational changes required for membrane fusion and viral entryinto the host cell. At present, efforts to develop lectins such asCV-N for therapeutic use are focused on topical applications inmicrobicides. All known anti-HIV lectins vary in their degreesof potency and some were found to have mitogenic activity (15).Therefore, the continued search for and characterization ofnovel HIV inhibitory lectins are important for moving micro-bicide development forward (16 –18). For CV-N, the presenceof two binding sites and the multivalency of the carbohydratehave been implicated as important factors for its anti-HIVactivity (6). Unfortunately, cross-linking-mediated aggregationand precipitation have hampered studies of CV-N/Man-9interactions at the atomic level in vitro (7, 19, 20) and haveprecluded unambiguous determination of whether multivalentand multisite sugar/protein interactions are a necessary prereq-uisite for the antiviral activity of cyanovirin-N homolog(CVNH) proteins. However, in contrast, considerable atomiclevel information is available on CV-N binding to substructuresof Man-8 and Man-9.

To elucidate the structural and mechanistic basis for the dif-ference between CV-N and Cyt-CVNH, we determined theCyt-CVNH crystal structure and assessed Man-2, Man-3, and

* This work was supported by National Institutes of Health grantRO1GM080642 (to A. M. G.). The authors declare that they have no conflictsof interest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official viewsof the National Institutes of Health.

□S This article contains supplemental Figs. S1–S4.The atomic coordinates and structure factors (code 5K79) have been deposited in

the Protein Data Bank (http://wwpdb.org/).1 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; E-mail:[email protected].

2 The abbreviations used are: CV-N, cyanovirin-N; CVNH, cyanovirin-N homo-log; ASU, asymmetric unit; PDB, Protein Data Bank; HSQC, heteronuclearsingle quantum coherence.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 36, pp. 18967–18976, September 2, 2016

Published in the U.S.A.

SEPTEMBER 2, 2016 • VOLUME 291 • NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 18967

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Man-9 binding by solution NMR. The structure of Cyt-CVNHis similar to that of other members of the CVNH family, alsopossessing two carbohydrate-binding sites, one per domain.However, in contrast to CV-N, no cross-linking and aggrega-tion is observed in the interaction with Man-9, permitting, forthe first time, determination of accurate affinities for Man-9binding to a CVNH lectin.

Cyt-CVNH inhibits HIV-1 in the low nanomolar concentra-tion range and possesses �4-fold higher potency than CV-N.Based on these structural and functional results, we suggest thatCyt-CVNH holds significant promise for future clinicalapplications.

Results

Crystal Structure of Cyt-CVNH—Here, we report the crystalstructure of a new CVNH, Cyt-CVNH, a recently identifiedlectin from the cyanobacterium Cyanothece7424 (21), whichcomprises 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. Thegenome shows that these cells have the ability to store the prod-ucts of both photosynthesis (glycogen) and nitrogen fixation(cyanophycin) as intracellular inclusion bodies (21). Beyondthese basic findings, however, the precise role of Cyt-CVNHwithin the host is unknown at present.

The crystal structure of Cyt-CVNH was solved at 1.6 Å res-olution by molecular replacement for orthorhombic crystals inspace 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. TheNMR solution structure of wild type CV-N (PDB accessioncode 2EZM) (5) was used as the search model. All pertinentcrystallographic statistics are provided in Table 1. Two inde-pendent monomers in close proximity were selected as the ASUand are shown in Fig. 1A. Each independent monomer in theASU is also related to a copy of the other by pseudotranslationalnon-crystallographic symmetry. This relation, however, cannotbecome crystallographic by any alternative unit cell/symmetry

FIGURE 1. Crystal structure of Cyt-CVNH. A, left panel, ribbon representation of the Cyt-CVNH crystal structure, illustrating the relative orientation of the twomonomers (m1 and m2) in the asymmetric unit. The long loop (Leu68–Thr72) of Cyt-CVNH is colored in magenta. Center panel, view of the �A-weighted(2mFo � DFc) electron density map for the loop region Trp50–Asn54, contoured at 0.5 �. 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 thepseudo-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 betweenadjacent 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 monomersin 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.

Structure and Anti-HIV Activity of Cyt-CVNH

18968 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 36 • SEPTEMBER 2, 2016


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definition. The current structure of Cyt-CVNH is similar topreviously determined crystal structures of monomeric, non-domain-swapped CV-N variants (22, 23), in contrast to thedomain-swapped wild type CV-N structures (13, 24, 25). Weascertained that the x-ray data were incompatible with adomain-swapped dimer structure by omitting the hinge-loopregion (Trp49–Asn53) in the model employed for molecularreplacement. After the first refinement step, the electron den-sity map clearly showed strong density for the omitted region,connecting the two separate starting segments in a contiguouspolypeptide chain (Fig. 1A, middle panel).

Within the asymmetric unit, the monomers are orientedwith respect to each other by an angle of �104° between thelong axes of the two domains (AB�, A�B), using the S� atoms ofthe cysteines in the two disulfide bonds (Cys8/Cys59�;Cys59/Cys8�) to define the axes. This spatial arrangement is oppositein orientation to what was previously observed between the twohalves of the domain-swapped CV-N structures (�101°) in thetrigonal crystal (P3221; PDB accession code 3EZM; Fig. 1A,right panel).

A surface view of the two molecules in the ASU is provided inFig. 1B (left panel). The monomer-monomer interface involvesa different region, with Phe69 of one monomeric unit engaged ina crystal contact with Trp50 of the adjacent monomeric unit(Fig. 1B, middle panel). The side chain of Asp45 in monomer 1hydrogen bonds to the side chain of Lys6 in monomer 2, andsimilarly, the backbone carbonyl oxygen of Thr47 in monomer 1hydrogen bonds with the side chain amide group of Gln3 inmonomer 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 proteinas a function of sugar addition. Titration experiments were car-ried out with Man-2, Man-3, and Man-9. Interestingly, bindingis in slow exchange on the chemical shift scale at 298 K, which isdifferent from what was observed with wild type CV-N andMan2/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. Map-ping of the affected amide resonances clearly revealed that forall three sugars two binding sites exist: one on domain A andone on domain B.

A superposition of the spectra of Cyt-CVNH in the absence(black contours) and presence (cyan contours) of 15 molarequivalents of Man-2 are provided in Fig. 2A. Likewise, super-position of free Cyt-CVNH (black contours) and protein in thepresence of 6 molar equivalents of Man-3 or 2 molar equiva-lents of Man-9 (magenta) are provided in Fig. 2 (B and C,respectively). The extracted binding isotherms derived fromthe intensity changes of sugar-bound amide resonances of res-idues from domain A and B are depicted in the right panels. Thedissociation constants for Man-2 binding to the sites on domainA and B are 53.2 � 8.7 and 48.4 � 9.5 �M, respectively. Theequivalent Kd values for Man-3 are 7.5 � 1.2 and 9.4 � 0.8 �M,respectively.

In the past, attempts to structurally monitor Man-9 bindingto CV-N by NMR were hampered by extreme line broadeningand ultimately disappearance of resonances in the 1H-15NHSQC spectra, accompanied by precipitation of the sugar�protein caused by multisite/multivalent cross-linking (7, 19,20). In contrast to the findings with CV-N, no aggregation orprecipitation was observed for Man-9 binding to Cyt-CVNH.Therefore, it was possible to identify those amide resonancesthat were affected by Man-9 binding. Again, two binding sitesare present, and Kd values of �500 nM were obtained fordomain A and B. Thus, domain A and domain B of Cyt-CVNHpossess essentially the same affinities for Man-9. The sameholds for Man-2 or Man-3.

Because the D1 and D3 arms of Man-9 contain �132-linkedmannoses, a single molecule of Man-9 can interact with morethan one lectin molecule that recognizes Man�(1–2)Man�units. The glycan binding site of CV-N in domain A exhibits aslight preference for Man-3, whereas domain B preferentiallybinds Man-2, resulting in cross-linking when interacting withMan-9 (7, 19, 20). Thus, Man-9 interacts with CV-N with theD1 arm, engaging domain A, and the D3 arm, binding todomain B. For Cyt-CVNH, we noticed in the titration thatMan-9 binding elicited changes in the 1H-15N HSQC spectrumvery similar to those of Man-3 and somewhat different fromMan-2 (Fig. 3). For instance, at a 0.5:1 Man-2:Cyt-CVNH molarratio the Thr58 resonance (domain B) is shifted, whereas theThr7 resonance (domain A) is not affected (Fig. 3A). This isdifferent for Man-3 and Man-9 binding, where the Thr7 reso-nance (domain A) is affected, but not the Thr58 resonance (Fig.3, B and C). Furthermore, at saturation, i.e. when both bindingsites on the protein are sugar-bound, the pattern observed forthe Man-3 and Man-9 shifted resonances is very similar for theThr7 resonance (Fig. 3, B and C, right panels) and distinctlydifferent from the one observed upon Man-2 binding (Fig. 3A,

TABLE 1Data collection and refinement statistics for the x-ray structure

Cyt-CVNHa

Data collectionSpace group P21212Cell dimensions

a, b, c (Å) 93.8, 74.4, 36.5�, �, � (°) 90, 90, 90

Resolution (Å) 34.58–1.6ASU content (molecules) 2Rmerge 0.11(0.83)b

I/�I 23.6 (1.7)Completeness (%) 99.8 (95.6)Redundancy 13.1 (4.6)CC 1/2 0.98 (0.91)

RefinementResolution (Å) 34.58–1.6No. reflections 34,474Rwork/Rfree 20.2/23.3No. atoms 1,912

Protein 1,677Diethylene glycol 18Water 217

Average B value (Å2) 20.5Protein 18.0Diethylene glycol 34.1Water 38.7

Root mean square deviationsBond lengths (Å) 0.006Bond angles (°) 1.090

Ramachandran statisticsResidues in favorable regions (%) 98.1Residues 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.




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FIGURE 2. Carbohydrate binding by Cyt-CVNH. A, superposition of the 1H-15N HSQC spectra of free Cyt-CVNH (150 �M; black) and in the presence of 15-foldmolar excess of Man�(1–2)Man (cyan). B, free Cyt-CVNH (50 �M; black) and in the presence of 6-fold molar excess Man�(1–2)Man�(1–2)Man (magenta). C, freeCyt-CVNH (10 �M; black) and in the presence of 2-fold molar excess Man9GlcNAc2 (magenta). Resonances of residues that undergo chemical shift changesupon carbohydrate binding are labeled in A–C. For Man�(1–2)Man, Man�(1–2)Man�(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 molarratio) for each domain are shown. In the right panel of C, the chemical structure of Man9GlcNAc2 is shown. The D1 arm (magenta) contains the Man�(1–2)Man�(1–2)Man trimannoside, whereas the D3 arm (cyan) contains the Man�(1–2)Man dimannoside.




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right panel). This similarity between the Man-3 and Man-9effects is observed throughout the entire titration, for reso-nances belonging to both domains A and B (supplemental Figs.S1 and S2). For instance, at a 1:1 sugar-protein molar ratio, theThr86 resonance (domain B) clearly shows a smaller chemicalshift difference between free and sugar-bound protein forMan-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-9interact in similar fashion with domain A and domain B ofCyt-CVNH. At a 1:1 molar ratio of sugar:protein, amide reso-nances of the majority of residues in the binding site of domainA, including Gly2, Gln3, Thr7, Thr29, Leu30, Gln23, Asp102,Gly103, and Thr104 undergo chemical shift changes only uponMan-3 and Man-9 binding, whereas two residues, Phe4 andLys24, exhibit perturbed resonances for all three sugars (Man-2,Man-3, and Man-9). Similarly, resonances of residues indomain B, such as Gly46, Arg81, Asp83, and Thr86 undergochemical shift changes upon Man-3 and Man-9 binding at 1:1molar 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-2binding.

This is illustrated diagrammatically in the scheme ofMan-9 interacting with domains A and B of Cyt-CVNH (Fig.3D). In this scheme, Man-9 interacts with domains A and Bof Cyt-CVNH through its D1 arm only (Fig. 3D andsupplemental Figs. S3 and S4A), and it is likely that this typeof recognition, which is different from what is observed forCV-N (supplemental Fig. S4B), prevents cross-linking ofCyt-CVNH by Man-9.

To corroborate that indeed the Man9�Cyt-CVNH complexcomprises one protein and two sugars, we carried out NMRrelaxation 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, respec-tively. These values are consistent with the expected mass of�12 kDa for the protein and with the �16 kDa mass matchingthe 2:1 Man-9�Cyt-CVNH complex (26).

HIV Assays—To determine the antiviral potency of the newlectin, Cyt-CVNH was assessed in parallel with monomeric

FIGURE 3. Oligomannose-9 binding to Cyt-CVNH. A–C, selected region of the superimposed 1H-15N HSQC spectra, showing the resonances of Thr7 (domain A) andThr58 (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, schematicillustration 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 (site2) are colored green and pink, respectively. The individual sugar units of the oligosaccharide are color-coded according to their linkage pattern.




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CV-NP51G, a stabilized version of wild type CV-N (25), in asingle-cycle luciferase reporter assay. This assay relies on com-pletion of the early steps in infection, including entry, reversetranscription, integration, and expression of the viral Tatprotein.

Normalized inhibition curves for concentrations up to 100nM 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.

Discussion

The envelope glycoprotein gp120 of HIV-1 mediates host cellentry, 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 ofthe viral and cellular membranes (27–29). Gp120 is highly gly-cosylated, containing a large number of high mannose sugars(8, 9, 30, 31), and the glycosylation sites are well conserved (32).A number of broadly neutralizing antibodies (Abs) target gly-cans on gp120 (33–35) and potentially could be used to combatHIV-1 infection. 2G12 was one of the first such antibodiesdescribed (36 –38), followed more recently by a collection ofAbs, including PGT121, PGT122, PGT128, and PGT135, thatrecognize dual protein-glycan epitopes, especially involving thesugar on Asn332 (33, 39 – 42). Crystal structures of glycosylatedsimian immunodeficiency virus gp120 (Ref. 43; PDB accessioncode 2BF1), as well as a HIV-1 trimer, complexed with thePGT122 antibody (Ref. 44; PDB accession code 4CNO) areavailable. Importantly, the Asn332 glycan is 73% conservedamong HIV isolates. The crystal structure of the gp120 trimerclearly delineates the Asn332 (Man8/9GlcNAc2) glycan as a keyelement in PGT122 recognition (39, 44).

Akin to mannose-targeting Abs, lectins also interact withsugars, and several mannose-targeting lectins have been shownto possess virucidal properties against HIV. CV-N, discoveredas one of the first HIV-1-inactivating lectins, is active againstHIV-1 and -2, simian immunodeficiency virus, and other envel-oped viruses like Ebola and Influenza (5) at nanomolar concen-

trations (6). Previous biochemical and biophysical studiesrevealed that two binding sites for Man�(1–2)Man�-contain-ing sugars are located on CV-N: one each on domains A and B.It was shown that CV-N recognizes the terminal Man�(1–2)Man� units of both the D1 and the D3 arms of Man-8 andMan-9 as the primary target (10 –14). Likewise, members of theCVNH family from Tuber borchii (TbCVNH), Ceratopterisrichardii (CrCVNH), Neurospora crassa (NcCVNH), and Gib-berella zeae (GzCVNH) also recognize Man�(1–2)Man disac-charides, but with lower affinity than CV-N (45, 46). Each ofthese proteins exhibits carbohydrate binding sites that are dif-ferent in number and location. CrCVNH possesses two sites;TbCVNH possesses a single binding site on domain A, whereasGzCVNH and NcCVNH have one site only on domain B. Withthe exception of GzCVNH, carbohydrate binding specificitiesare distinct as well, and no potent HIV inactivation wasobserved with any of these proteins (45, 46).

Although in principle very powerful for delineating bindingsites on proteins, using 1H-15N HSQC spectroscopy for follow-ing Man-9 binding to CV-N, even at low concentration, wasimpossible because precipitation of the sugar�protein complexoccurred, caused by multisite/multivalent cross-linking (7, 19,20). Here, by contrast, for Cyt-CVNH, no aggregation or pre-cipitation was seen for the Man9�Cyt-CVNH complex. In addi-tion, based on the patterns observed throughout the titrationswith Man-2, Man-3, and Man-9, it is evident that Man-9 bindsboth sites on Cyt-CVNH through its D1 arm only (Fig. 3D).Thus, unlike for CV-N, where 1:1 binding between Man-9 andprotein is causing precipitation, for Cyt-CVNH a 2:1sugar�protein complex is formed, without any cross-linkedhigher molecular species noted.

Based on all available data, we developed an interactionmodel for CVNH lectins and glycosylated gp120, assuming thatthe interaction involves the glycan on Asn332. As the startingmodel, we used the crystal structure of the HIV-1 Env trimer incomplex with the antibody PGT122 (blue; PDB accession code4NCO; the antibody coordinates are omitted from one of thegp120 monomeric units). Onto the trimer, we superimposedthe monomeric gp120 core structure (orange) in complex withCD4 (magenta; PDB accession code 3JWD) (Fig. 5, top panel).As can be appreciated, the Env trimer structure contains a largenumber of sugar molecules. In addition to Asn332, which bearsMan8/Man9, the PGT122 Ab interacts with the base of the V1and V3 loops on the protein and three more glycans on Asn301,Asn156/Asn173, and Asn137 (44).

The distance between the two glycan binding sites on CVNHlectins is �40 Å. If the D1 arm of Man-8/Man-9 on Asn332 isbinding one of these two sites, a possible second sugar onAsn156 could interact with the second site. First, we placed theCyt-CVNH x-ray structure onto the Asn332 glycosylation site.This allows Man8/Man-9 to interact with domain A. Next werotated the Cyt-CVNH model around the Asn332 sugar, to findanother possible sugar on gp120 that could interact withdomain B; this was the sugar on Asn156. Using the sugars onAsn156 and Asn332 (Fig. 5, bottom panel), the CVNH lectin iscontacting approximately the same region as the PGT122antibody.

FIGURE 4. Anti-HIV activity. Single-cycle HIV-1 infectivity assays carried outwith Cyt-CVNH (orange-filled circles) and CV-NP51G (black open circles), using aluciferase reporter cell line. The symbols are experimental data points with thebest fit to the data shown as continuous lines.




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It is well established that interaction of CD4 with the HIVenvelope causes conformational changes in the trimer, leadingto a more open conformation that exposes the co-receptorbinding site (47). Although steric occlusion of the CD4 bindingsite by PGT121 binding to gp120 does not seem to be the mech-anism for competitive inhibition, allosteric effects that interferewith the CD4 induced conformational changes may play a role(40). Such mechanisms may also apply to glycan-binding lec-tins, although other mechanisms cannot be ruled out at pres-ent, and further studies are necessary to determine whetherCVNH lectins are capable of preventing CD4 binding throughan allosteric mechanism. Alternatively, they may exert theirantiviral activity by inducing post-binding conformationaleffects that prevent CD4-bound gp120 from interacting withCCR5 or CXCR4 co-receptors.

Experimental Procedures

Protein Expression and Purification—The protein wasexpressed from a synthetic gene, using pET26b(�) (Novagen;Madison, WI) and Escherichia coli BL21(DE3) as expressionvector and host strain, respectively. The cells were initially

grown at 37 °C, induced with 1 mM isopropyl �-D-thiogalacto-pyranoside at 16 °C, and grown for �12 h at 16 °C for proteinexpression. Uniform 15N and 13C labeling of Cyt-CVNH wascarried out by growth in modified minimal medium, using15NH4Cl and 13C6-glucose as the sole nitrogen and carbonsources, respectively. The cells were harvested by centrifuga-tion (4600 g for 15 min at 4 °C), resuspended in 20 mM potas-sium phosphate buffer, pH 6.0, and lysed using a mircrofluid-izer (MicroFluidics M-110Y, Hyland Scientific). Cell debris wasremoved by ultracentrifugation (120,000 g), and the super-natant was fractionated by gel filtration on a Superdex 75(HiLoad 2.6 60 cm; Amersham Biosciences) column, equili-brated in 20 mM sodium phosphate buffer, pH 6.0. Protein frac-tions containing Cyt-CVNH were collected and concentratedup to 10 mg/ml using Centriprep devices (Millipore). Pro-tein purity was estimated 99% by SDS-PAGE and massspectrometry.

Crystallization and X-ray Data Collection—Cyt-CVNH pro-tein was crystallized by sitting drop vapor diffusion from a 1.0mM protein solution in 20 mM sodium phosphate buffer, 0.01%NaN3, pH 6.0. The best crystals were obtained at room temper-ature 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 asprecipitants. Crystal growth took �30 days, yielding crystalswith dimensions of 0.20 0.30 0.70 mm. X-ray diffractiondata were collected from a single flash-cooled crystal (�180 °C)at the Southeast Regional Collaborative Access Team facilitysector 22-ID beam line of the Advance Photon Source(Argonne National Laboratory, Chicago, IL). 451,609 totalobservations 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 datawere processed and scaled with the HKL2000 package (48).

Crystal Structure Determination and Refinement—The crys-tal structure of Cyt-CVNH was solved by molecular replace-ment in Phenix (49), using the monomeric NMR structure ofwild type CV-N (PDB accession code 2EZM) (5) as the searchmodel. The initial model included two independent segmentsof the chain, comprising residues Leu1–Lys48 and Phe54–Glu101, with the hinge-loop region (Trp49–Asn53) omitted. Iter-ative rigid body and simulated annealing refinement in Phenixwas alternated with model building, including the hinge-loopregion, in Coot (50). The final stages of refinement includedperiodic examinations of �A-weighted electron density (2mFo �DFc) and difference electron density (mFo � DFc) maps, as wellas the introduction of water and several cryoprotectant mole-cules. Analysis of the final structural model was performedusing PROCHECK (51). Approximately 98% of all residuesreside in the favored region of the Ramachandran plot (52) withno residues in the disallowed regions. The final model was alsovalidated by MolProbity (53), with an overall clash score of 0.31and percentile of 100%. Atomic coordinates and structurefactors have been deposited in the Research Collaboratoryfor Structural Bioinformatics Protein Data Bank underaccession code 5K79. All structural figures were generatedwith PyMOL (54).

NMR Spectroscopy—NMR spectra were recorded at 298 K onBruker 600 MHz and 800 MHz AVANCE spectrometers,

FIGURE 5. Model for the interaction between CVNH lectins and glycosy-lated 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 trimeris shown in yellow. Gp120 glycosylation sites are labeled on one monomer,and the sugars are shown in blue stick representation. The x-ray structure ofCyt-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 sugaron Asn332 and Asn156 and Cyt-CVNH. The epitope used by the lectin is locatedon the opposite side of the CD4 binding site, similar to where PGT122 isbound.




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equipped with 5 mm, triple resonance, three-axis gradientprobes, or z axis gradient cryoprobes. For three-dimensionalNMR experiments, the sample contained 0.5 mM protein in 20mM sodium phosphate buffer, pH 6.0. For chemical shift assign-ments, a series of heteronuclear, multidimensional experi-ments, routinely used in our laboratory, were recorded (55–58).Complete 1H, 15N, and 13C backbone resonance assignmentswere obtained using NMR data obtained from two-dimensional1H-15N HSQC, three-dimensional HNCACB, HN(CO)CACB,HNCA, and HN(CO)CA spectra.

Binding Studies—Binding of sugar to protein was assessed intitration experiments, using uniformly 15N-labeled Cyt-CVNH(150 �M) at 298 K in 20 mM sodium phosphate buffer, pH 6.0,0.01% sodium azide, and 90% H2O/10% D2O, monitoring thechemical shift changes in 1H-15N HSQC spectra upon sugaraddition. For dimannose (Man-2) binding, aliquots of a 50 mM

Man-2 stock solution were added to yield sugar/protein molarratios of: 0, 0.5, 1, 2, 3, 4, 5, 7, 10, and 15. Analogous NMRtitration experiments were performed with trimannose (Man-3), using aliquots of a 10 mM stock solution to yield sugar/pro-tein molar ratios of: 0, 0.5, 1, 2, 3, 4, 5, and 6. The proteinconcentration in the titrations with oligomannose-9 (Man-9)was 10 �M, and aliquots from a 500 �M stock solution wereadded to yield sugar/protein molar ratios of: 0, 0.5, 1, and 2.

Free and sugar-bound protein resonances are in slowexchange on the chemical shift scale for all three sugars. Theobserved signal intensity of the sugar-bound protein reso-nances during the titration is directly proportional to the boundfraction (fb) and is given as fb � Ib/Ib max � [PL]/[P], where [P] isthe total concentration of protein, [PL] is the concentration ofthe protein�ligand complex, Ib is the intensity of the sugar-bound protein signal at each point in the titration, and Ib max isthe maximum sugar-bound protein signal intensity at the endof 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 bynonlinear best fitting of the titration curves using KaleidaGraph(Synergy Software, Reading, PA) and the following equation:

fb � Ib/Ib max � 0.5*�M � 1 �KD

�P� ��M � 1 �

KD

�P��2

4M �(Eq. 1)

Anti-HIV Assay—HIV-1 infectivity was assayed as describedpreviously (59). For antiviral assays, recombinant proteins wereserially diluted in sterile phosphate-buffered saline, and 5 �lwere added to 500 �l of prediluted infectious HIV-1 (producedby transfection of 293T cells with the R9 molecular clone andincubated for 30 min at room temperature). Aliquots of themixture (125 �l, in duplicate) were added to cultures of TZM-blcells (20,000 cells seeded per well the day before in a 48-wellformat), and after 2 days, cells were lysed and assayed for lucif-erase activity as previously described (60). IC50 values weredetermined by nonlinear best fitting of the normalized inhibi-tion curves using KaleidaGraph (Synergy Software, Reading,PA). The results are representative of two independentexperiments.

Author Contributions—E. M. and A. M. G. conceived and coordi-nated the study. R. B. performed NMR assignments and helped withNMR titrations and the preparation of figures, C. C. performed crys-tal optimization, and W. F. collected the diffraction data at theAdvance Photon Source and was involved in x-ray data interpreta-tion. 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 supportand Palaniappa Arjunan for help with x-ray diffraction data collec-tion at the Advance Photon Source.

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and Angela M. GronenbornElena Matei, Rohan Basu, William Furey, Jiong Shi, Conor Calnan, Christopher Aiken

Structure and Glycan Binding of a New Cyanovirin-N Homolog

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