Arulanandam, Jeyaprakash

Kenoth, R

Komath, S S

Manoj, N

Pratap, J V

Swamy, M J

Vijayan, M

English

The lectin from the seeds of snake gourd (Trichosanthes anguina) has been crystallized in two forms using the hanging-drop method. Both the forms are hexagonal, with the asymmetric unit containing one subunit consisting of two polypeptide chains linked through disulfide bridges. Intensity data from one of the forms were collected at room temperature as well as at low temperature to 3 Angstrom resolution. Molecular-replacement studies indicate that the lectin is homologous to type II ribosome-inactivating proteins. Partial refinement confirms this conclusion.</p

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     Edinburgh Research Explorer                                      Crystallization and preliminary X-ray studies of snake gourdlectin: homology with type II ribosome-inactivating proteinsCitation for published version:Manoj, N, Arulanandam, J, Pratap, JV, Komath, SS, Kenoth, R, Swamy, MJ & Vijayan, M 2001,'Crystallization and preliminary X-ray studies of snake gourd lectin: homology with type II ribosome-inactivating proteins' Acta Crystallographica Section D-Biological Crystallography, vol 57, pp. 912-914.,10.1107/S0907444901004620Digital Object Identifier (DOI):10.1107/S0907444901004620Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher final version (usually the publisher pdf)Published In:Acta Crystallographica Section D-Biological CrystallographyPublisher Rights Statement:RoMEO greenGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 20. Feb. 2015912 Manoj et al.  Snake gourd lectin Acta Cryst. (2001). D57, 912±914crystallization papersActa Crystallographica Section DBiologicalCrystallographyISSN 0907-4449Crystallization and preliminary X-ray studies ofsnake gourd lectin: homology with type IIribosome-inactivating proteinsN. Manoj,a A. ArockiaJeyaprakash,a J. V. Pratap,aSneha Sudha Komath,b RoopaKenoth,b Musti. J. Swamyb andM. Vijayana*aMolecular Biophysics Unit, Indian Institute ofScience, Bangalore 560 012, India, and bSchoolof Chemistry, University of Hyderabad,Hyderabad 500 046, IndiaCorrespondence e-mail: mv@mbu.iisc.ernet.in# 2001 International Union of CrystallographyPrinted in Denmark ± all rights reservedThe lectin from the seeds of snake gourd (Trichosanthes anguina) hasbeen crystallized in two forms using the hanging-drop method. Boththe forms are hexagonal, with the asymmetric unit containing onesubunit consisting of two polypeptide chains linked through disul®debridges. Intensity data from one of the forms were collected at roomtemperature as well as at low temperature to 3 AÊ resolution.Molecular-replacement studies indicate that the lectin is homologousto type II ribosome-inactivating proteins. Partial re®nement con®rmsthis conclusion.Received 17 January 2001Accepted 14 March 20011. IntroductionLectins are carbohydrate-binding proteins thatspeci®cally recognize diverse sugar structuresand mediate a variety of biological processessuch as cell±cell and host±pathogen inter-actions, serum glycoprotein turnover andinnate immune responses (Lis & Sharon, 1998;Vijayan & Chandra, 1999). Of them, plantlectins are the most extensively and thoroughlycharacterized (Lis & Sharon, 1998; Loris et al.,1998; Bouckaert et al., 1999). The differentfamilies, well characterized structurally,include one involving plant toxins. Ricin(Rutenber et al., 1991), abrin (Tahirov et al.,1995) and mistletoe lectin (Krauspenhaar et al.,1999) are lectins of known structure belongingto this family. Each of them typically consists oftwo polypeptide chains, each with a well char-acterized structure and often referred to as Aand B domains, connected by disul®de bridges.They are often called type II ribosome-inactivating proteins (type II RIPs). The Achain is the toxic component and inactivatesribosome. The B chain is the lectin componentand is presumably involved in recognition priorto entry into the cell. Type I ribosome in-activating proteins (type I RIPs) such asgelonin (Sairam et al., 1993; Singh et al., 1992),trichosanthin (Xiong et al., 1994) andmomorcharin (Ren et al., 1994) consist only ofthe A domain, while there are lectins such asamaranthin (Transue et al., 1997) which consistonly of the B domain. The latter obviously donot have any ribosome-inactivating activity.However, as indicated earlier, type II RIPscombine toxic and lectin activities on accountof the simultaneous presence of the A and Bdomains.Macromolecular and carbohydrate-bindingproperties of several seed lectins from theCucurbitaceae family have been investigated(Falasca et al., 1989; Bostwick et al., 1994;Komath & Swamy, 1998; Padma et al., 1999).Detailed structural characterization of any ofthese has not been possible on account of,amongst other things, the non-availability oftheir complete amino-acid sequence data.Various lines of evidence suggest that they arestructurally homologous to type II RIPs.However, they do not inactivate ribosomes ordo so only weakly (Wang & Ng, 1998; Li et al.,2000). The X-ray analysis of the seed lectinfrom T. anguina, a member of the Cucurbita-ceae family commonly known as snake gourd,has been undertaken to elucidate the structuralbasis of this behaviour and also to explore thestructure±function relationship in this family oflectins. Snake gourd seed lectin (SGSL) isglycosylated and has an acidic pI of 5.0. Thelectin displays haemagglutinating activity andshows a high af®nity for methyl--d-galactoseat the monosaccharide level and for lactose atthe disaccharide level (Komath & Swamy,1998). It contains two domains, MW = 32 and23 kDa, which are linked by disul®de bridges(Komath et al., 1996). Chemical-modi®cationstudies indicated the presence of histidineresidues in the sugar-binding site of SGSL(Komath et al., 1998). The lectin has bindingsites for porphyrins which appear to bedifferent from those for carbohydrates(Komath et al., 2000). As a ®rst step in thedetailed structure analysis of SGSL, we reporthere its crystallization and structure solutionusing molecular replacement and partialre®nement of the structure.2. Materials and methodsSGSL was isolated and puri®ed as describedpreviously (Komath et al., 1996; Komath &Swamy, 1998). Crystals of two forms of SGSL,form I and form II, were grown in the presenceof lactose and methyl--d-galactose, respec-Acta Cryst. (2001). D57, 912±914 Manoj et al.  Snake gourd lectin 913crystallization paperstively. Crystal form I was obtained by thehanging-drop method by equilibrating a10 ml drop of 10 mg mlÿ1 protein in thepresence of 4 mM lactose in 0.02 M phos-phate buffer pH 7.0 containing 0.1 M NaCland 0.025%(w/v) sodium azideagainst a reservoir solution of 80%saturated ammonium sulfate inthe same buffer. The crystals grewto ®nal dimensions of 0.4  0.2 0.08 mm in 60 d. Crystals of formII were also grown similarly(Fig. 1). The drop contained 10 mlof 40 mg mlÿ1 protein in thepresence of 10 mM methyl--d-galactose, 5 mM -mercapto-ethanol and 1 ml of 30% PEG 400.The reservoir solution contained1 ml of 80% saturated ammoniumsulfate in the same buffer. Thesecrystals grew to maximum dimen-sions of 1.5  1.0  0.3 mm inabout 45 d. Crystal form Idiffracted to about 6 AÊ , whileform II diffracted to 3 AÊ resolu-tion. Diffraction data from form IIwere collected at room tempera-ture (298 K) and at low temperature (100 K)using ¯ash-freezing with a MAR Researchimaging plate mounted on a Rigaku RU-200X-ray generator. The data were processedand scaled using the MAR-XDS (Kabsch,1988) and DENZO (Otwinowski, 1993)programs. AMoRe (Navaza, 1994) was usedfor molecular-replacement calculations,while preliminary re®nement and modelbuilding were carried out using the programsCNS (Brunger et al., 1998) and O (Jones etal., 1991), respectively.3. Results and discussionCrystal data and details of data collectionare given in Table 1. The solvent contents ofboth forms, assuming one molecule in theasymmetric unit, are within the rangenormally observed for protein crystals(Matthews, 1968).Unlike the case of type II RIPs, theamino-acid sequences of type I RIPs fromseeds of Momordica charantia and Tricho-santhes kirilowii belonging to the Cucurbi-taceae family are available. They exhibitconsiderable sequence similarity to eachother and with well studied type I RIPs fromthe Cucurbitaceae family such as bryodinand luf®n and the A chain of type II RIPssuch as abrin, ricin and mistletoe lectin.Therefore, it appeared reasonable to expectthe A chain of SGSL also to be homologousto the type II RIPs mentioned above. Therecent preliminary X-ray studies andmolecular-replacement calculation of thelectin from T. kirilowii strengthened thisexpectation (Li et al., 2000). In con®rmation,a molecular-replacement calculation withabrin-a (Tahirov et al., 1995; PDB code 1abr)as the search model using the low-temperature data from form II led tosatisfactory structure solution. A long loop(41±50) in the A chain and ten residues atthe N-terminus of the B chain were omittedfrom the search model. The best solutionhad a correlation coef®cient (CC) of 0.345and an R factor of 0.489 and led to satis-factory crystal packing. The correspondingnext best solution had CC = 0.289 andR = 0.504. Similar results were obtainedwhen the ricin molecule (PDB code 1aai)was used as the search model. Independentcalculations using the A chain and B chain assearch models also led to the same solution.A Fourier map calculated after rigid-bodyre®nement using a polyalanine modelshowed contiguous density for most of thepolypeptide chains. This was particularly soin the case of the A chain.Further re®nement of the structure washampered by the absence of sequenceinformation. However, it was found that theA chain of type II RIPs such as ricin andabrin and type I RIPs from the Cucurbita-ceae family exhibited considerable sequencehomology. Thus, it appeared that thesequence of the A chain is substantiallyconserved across families. Sequence identityamong the Cucurbitaceae type I RIPsequences is as high as 52%; there is also a38% sequence identity with ricin and abrinsequences. Careful examination of thesesequences and successive electron-densitymaps led to the identi®cation of 31% of theside chains in the A chain. This could not beperformed with the B chain in the absence ofthe availability of homologous sequencesfrom the same family. Re®nement of themodel using data in the resolution range 20±Figure 1Crystals of snake gourd seed lectin (form II).Table 1Crystal data and data-collection statistics.Values in parentheses refer to the last resolution shell.Form IIForm ILow temp.(100 K)Room temp.(298 K)Space group P622 P6122 P6122Unit-cell parametersa (AÊ ) 118.9 98.9 102.1b (AÊ ) 118.9 98.9 102.1c (AÊ ) 228.4 266.9 273.1No. of subunits/asym. unit 1 1 1Solvent content (%) 67 59 62Data collectionResolution (AÊ ) 6.0 3.0 (3.1±3.0) 3.1 (3.2±3.1)No. of observations Ð 65920 51684No. of unique re¯ections Ð 15685 14799Multiplicity Ð 4.2 (3.7) 3.5 (3.3)Completeness (%) Ð 98.0 (92.3) 92.7 (92.2)Rmerge² (%) Ð 12.1 (38.2) 16.0 (56.3)Number of re¯ectionswith I > 2(I) (%)Ð 85.9 (65.1) 64.8 (39.1)² Rmerge =P jIi ÿ hIij=PhIi.Figure 2Electron density in the putative active site in the A domain of the lectin. The relevant residues in the lectin and intrichosanthin are shown in red and blue, respectively. The numbering corresponds to that in trichosanthin. Nodensity corresponding to the side chain of Tyr70 in trichosanthin is seen in the map. Density for a side chain ispresent corresponding to Gly109.914 Manoj et al.  Snake gourd lectin Acta Cryst. (2001). D57, 912±914crystallization papers3.0 AÊ employing the `mlf' target wasperformed, resulting in an R factor of 0.34and an Rfree of 0.40 (with 8% omitted data).Further re®nement of the structure is inprogress. Sequence determination usingchemical means is also under way. However,the preliminary crystallographic results andthe available biochemical data indicate thatSGSL is homologous to type II RIPs. Ourstudies also provide a rationale for thestructure not possessing the same level ofribosome-inactivating activity as abrin-a andricin. The crystal structure of trichosanthin(TCS) complexed with a substrate analoguerevealed the role of conserved amino-acidresidues in the active-site cleft in theN-glycosidase activity of RIPs (Xiong et al.,1994). A comparison of our preliminarySGSL model with that of RIPs indicated thatthe fully conserved Tyr70 (TCS numbering)that is important for the biological activity isreplaced by a non-aromatic residue in SGSL(Fig. 2). Also, a conserved Gly109 appears tobe replaced by a larger residue. A similarresult was also obtained in preliminarystructural studies of Trichosanthes kirilowiilectin 1 (Li et al., 2000). A detailed analysisof the structure of SGSL should thus revealthe structural basis for the difference inbiological activity between SGSL and otherRIPs.The intensity data were collected at theX-ray Facility for Structural Biology at theInstitute, supported by the Department ofScience and Technology (DST) and theDepartment of Biotechnology (DBT).Facilities at the Supercomputer Educationand Research Centre and the InteractiveGraphics based facility and DistributedInformation Centre (both supported byDBT) were used in the work. The work atBangalore and Hyderabad was supported byResearch Grants from the DST to MV andthe Council of Scienti®c and IndustrialResearch (CSIR) to MJS.ReferencesBostwick, D. E., Skaggs, M. I. & Thompson, G. A.(1994). Plant Mol. Biol. 26, 887±897.Bouckaert, J., Hamelryck, T., Wyns, L. & Loris, R.(1999). Curr. Opin. Struct. Biol. 9, 572±577.Brunger, A. T., Adams, P. D., Clore, G. M., Delano,W. L., Gros, P., Grosse-Kunstleve, R. 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Crystallization and preliminary X-ray studies of snake gourd lectin: homology with type II ribosome-inactivating proteins

Crystallization and preliminary X-ray studies of snake gourd lectin: homology with type II ribosome-inactivating proteins

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