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Dimerization of a flocculent protein from Moringaoleifera: experimental evidence and in silicointerpretationAsalapuram R. Pavankumara, Rajarathinam Kayathrib, Natarajan A. Muruganb, Qiong Zhangb,Vaibhav Srivastavac, Chuka Okolia, Vincent Bulonec, Gunaratna K. Rajaraoa & Hans Ågrenb

a Division of Bioprocess Technology, School of Biotechnology, Royal Institute of Technology(KTH), 10691, Stockholm, Sweden.b Division of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute ofTechnology (KTH), 10691, Stockholm, Sweden.c Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH),10691, Stockholm, Sweden.Published online: 10 May 2013.

To cite this article: Asalapuram R. Pavankumar, Rajarathinam Kayathri, Natarajan A. Murugan, Qiong Zhang, VaibhavSrivastava, Chuka Okoli, Vincent Bulone, Gunaratna K. Rajarao & Hans Ågren (2014) Dimerization of a flocculent protein fromMoringa oleifera: experimental evidence and in silico interpretation, Journal of Biomolecular Structure and Dynamics, 32:3,406-415, DOI: 10.1080/07391102.2013.770374

To link to this article: http://dx.doi.org/10.1080/07391102.2013.770374

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Dimerization of a flocculent protein from Moringa oleifera: experimental evidenceand in silico interpretation

Asalapuram R. Pavankumaraz, Rajarathinam Kayathribz, Natarajan A. Muruganbz, Qiong Zhangb, Vaibhav Srivastavac,Chuka Okolia, Vincent Bulonec, Gunaratna K. Rajaraoa* and Hans Ågrenb*aDivision of Bioprocess Technology, School of Biotechnology, Royal Institute of Technology (KTH), 10691, Stockholm, Sweden;bDivision of Theoretical Chemistry and Biology, School of Biotechnology, Royal Institute of Technology (KTH), 10691, Stockholm,Sweden; cDivision of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), 10691, Stockholm, Sweden

Communicated by Ramaswamy H. Sarma.

(Received 20 January 2013; final version received 22 January 2013)

Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, thedimerization property of a coagulant protein (MO2.1) from Moringa oleifera seeds was addressed through laboratoryexperiments, protein–protein docking studies and binding free energy calculations. The structure of MO2.1 was predictedby homology modelling, while binding free energy and residues-distance profile analyses provided insight into the ener-getics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms ofMO2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.

Keywords: flocculent protein; oligomerization; homology modelling; binding free energy; protein–protein interactions

Introduction

The seed proteins of the tropical plant Moringa oleif-era (MO) possess a wide range of useful properties,including coagulation and antimicrobial activities(Anwar, Latif, Ashraf, & Galim, 2007; Chun-Yang,2010; Fahey, 2005; García-Fayos, Arnal, Verdú, & Rodr-igo, 2010). The crude seed powder has gained attentionas it could be a potential alternative for chemical usagein water treatment processes for the removal of heavymetal ions, dyes, industrial effluents and detergents (Belt-rán-Heredia & Sánchez-Martín, 2008; Beltrán-Heredia,Sánchez-Martín, & Delgado-Regalado, 2009). Amongthe seed proteins, a 6.5-kDa water-soluble coagulantprotein (MO2.1) has been extensively studied for itsapplications in water treatment (Broin et al., 2002;Ferreira et al., 2011; Gassenschmidt, Jany, Tauscher, &Niebergall, 1995; Ghebremicahel, Gunaratna, Henriks-son, Brumer, & Dalhammar, 2005; Ndabigengesere,Narasiah, & Talbot, 1995; Suarez et al., 2003). However,there are different opinions regarding its actual molecularmass: earlier reports have predicted a molecular massranging between 3.5 and 14 kDa (Gassenschmidt et al.,1995; Ndabigengesere et al., 1995; Okuda, Baes,

Nishijimam, & Okadam, 2001), whereas more recentreports suggested that the protein may consist of severalsubunits forming an overall molecular mass of 17–26 kDa (García-Fayos et al., 2010) or a single cationicprotein of 66 kDa (Agrawal, Shee, & Sharma, 2007).Moreover, the proposed mechanism of MO2.1 in watertreatment is unclear (Vlahov, Chepkwony, & Ndalut,2002). Having limited information about the biochemicaland structural properties of this protein (Kwaambwa &Maikokera, 2008; Kwaambwa & Rennie, 2012), it is dif-ficult to understand the corresponding molecular mecha-nisms. In this regard, one can speculate thatoligomerization of MO2.1 may be responsible for dis-playing differences in molecular masses, which mighthinder the elucidation of its fine structure and mechanismof action.

Oligomerization is a well-known phenomenon inmany cellular proteins and enzymes that refers to theself-association of polypeptide chains to form dimers orhigher order oligomers, thereby leading to a gain instructural and functional properties (Marianayagam,Sunde, & Matthews, 2004). Interestingly, some proteinslike the bovine (Bos Taurus) acetylgalactosaminidase is

*Corresponding authors. Email: agren@theochem.kth.se; gkr@kth.sezAuthors contributed equally.

Journal of Biomolecular Structure and Dynamics, 2014Vol. 32, No. 3, 406–415, http://dx.doi.org/10.1080/07391102.2013.770374

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non-functional in its monomeric form but becomesfunctional after oligomerization into a tetrameric form(Weissmann & Wang, 1971), which illustrates theimportance of oligomerization in proteins for acquiringfunctionality. In plants, nucleotide binding site–leucine-rich repeat proteins that are similar to the mammaliannucleotide-binding oligomerization domain proteinfamily and are dependent on oligomerization (ligandmediated) for functionality (Mestre & Baulcombe, 2006).Similarly, the function of an important enzyme, UDP-glucose pyrophosphorylase, which synthesizes the nucle-otide-sugar UDP-glucose as a precursor for saccharides,is also dependent on oligomerization and de-oligomeriza-tion (Martz, Wilczynska, & Kleczkowski, 2002). Yet, insome cases, oligomerization may become disadvanta-geous due to the formation of aggregates that lead tomalfunctioning of the proteins (Dobson, 2003; Ross &Poirier, 2003). Therefore, understanding the oligomeriza-tion process at the molecular level is important, as suchinsight might be useful to, for example, identify newtherapeutics to prevent protein aggregation in diseaseslike, for instance, the Alzheimer and Parkinson diseases(Dobson, 2003; Dobson, Ellis, & Fersht, 2001).

In order to understand the dimerization and oligomeri-zation of a protein, it is essential to obtain the structure ofthe monomer. Since the crystal structure of MO2.1 is yetto be solved experimentally, we have determined here aplausible tertiary structure based on homology modelling.Subsequently, a protein–protein docking study was per-formed to propose a structure for the dimer. Studyingoligomerization or self-association of a protein is oftenvery challenging due to the increased search space forvarious protein–protein binding modes, the number ofdegrees of freedom associated with protein oligomers andthe relatively large time scale associated with the self-association process, which is typically too large formolecular dynamics (MD) studies. In order to avoid suchlimitations of MD in modelling long time scale processes,the structure of oligomers or self-associated proteins wasinitially obtained through protein–protein docking studies.However, this provides only first-hand information aboutthe mode of binding and oligomer interaction energy. Toaccount for the contributions due to conformational sam-pling, binding free-energy calculations were performedbased on the molecular mechanics–Poisson Boltzmann(and Generalized Born) surface area approach for variousconfigurations obtained from MD simulations. Finally,protein residue contact analysis was performed to under-stand if residue contacts stabilize dimerization of the pro-tein. Simultaneously, coagulation activity was studiedwith a construct expressing the MO2.1 protein as a�14 kDa homodimer, as well as after reduction of therecombinant dimer to �6.5 kDa monomers. This wascompared with the protein profile and functional proper-ties of crude seed extracts of MO.

Materials and methods

Protein expression and profile analysis

The gene coding for MO2.1 was cloned and expressed inEscherichia coli TOPO® cells (Invitrogen), according tothe manufacturer’s instructions. To construct the clone,degenerate primers corresponding to segments of MO2.1

were used as described by Broin et al. (2002). TheE. coli clones were cultured in Luria Bertani broth andinduced with isopropyl β-D-1-thiogalactopyranoside(IPTG); the cells were harvested when the optical densityreached 0.8 at 600 nm. The recombinant MO2.1 proteinwas obtained from the sub-cellular fractions of E. coli asa homodimer (14 kDa), which was further reduced to itsmonomeric form with an optimal concentration of40mM dithiothreitol (DTT) in all the buffers, from lysisto elution (Klein, Lee, Frank, Marks, & Lemmon, 1998;Suarez et al., 2003). The whole-cell lystates were pre-pared according to Pavankumar, Ayyappasamy, andSankaran (2012) supplementing 40mM DTT. The MOseeds were purchased from Senegal, and proteins wereextracted according to Ghebremicahel et al. (2005) andfrozen until experimentation. Protein concentrations weremeasured using the Bradford dye-binding assay(Bradford, 1976) and �25 μg of total proteins wasanalysed on 10% SDS-PAGE and stained with Coomas-sie Brilliant Blue (CBB)-R250 (Laemmli, 1970).

In-gel digestion, mass spectrometry and data analysis

The CBB stained SDS-gel band corresponding to theMO2.1 protein was excised and digested in-gel asdescribed by Srivastava et al. (2009). The resulting pep-tides were analysed by reverse-phase LC-ESI-MS/MSusing a nanoACQUITY ultra-performance liquid chroma-tography system coupled to a quadruple time-of-flightmass spectrometer (Xevo Q-tof, Waters Corporation) indata-dependent mode. The ProteinLynx Global Serversoftware (V2.2.5) was used to convert raw data to peaklists for database searching, and the MO2.1 protein wasidentified by a local version of the Mascot search pro-gram (V2.3.1, Matrix Science Limited, http://www.matrixscience.com), using the Swiss-Prot database withtaxonomy “Viridiplantae” (green plants). The followingsettings were used for the database search: trypsin-specific digestion with one missed cleavage allowed,carbamidomethylated cysteine set as fixed modification,oxidized methionine in variable mode, peptide toleranceof 20 ppm and fragment tolerance of 0.1 Da. Peptideswith Mascot ion scores exceeding the threshold forstatistical significance of p< 0.05 were selected.

Coagulation activity analysis

Coagulation activities of the E. coli cell lysates treatedor not with DTT and of the crude MO seed extracts wereevaluated in 1-ml polystyrene cuvettes. Ten microlitres

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of each protein sample (1 μg/μl) was added to 990 μl of1% (w/v) synthetic turbid water. The absorbance wasmeasured at 500 nm at 30min time intervals for 90min,as described by Ghebremicahel et al. (2005). The per-centage of activity was calculated by the formula:

% of activity ¼ Initial Abs� Final Abs

Initial Abs� 100

Preliminary bioinformatic analysis of MO2.1

Preliminary information about MO2.1 was obtained fromvarious scientific reports and public databases, essentiallyNCBI, SwisProt and PDB. The polypeptide sequence ofMO2.1 originally published by Gassenschmidt et al.(1995; accession # P24303) was used to study the aminoacid sequence and their charges, sequence similarities,peptide mapping, proteolytic susceptibility. The prelimin-ary structure was analysed using ExPASy tools and thesequence obtained from NCBI and UniProt databases.

Homology modelling of coagulant protein

A BLAST search was performed using the MO2.1 poly-peptide sequence, QGPGRQPDFQRCGQQLRNISPPQR-CPSLRQAVQLTHQQQGQVGPQQVRQMYRVASNIPST(UniProt ID: P24303) against the PDB database to iden-tify a compatible structural homologue. The homologueof MO2.1 was obtained through homology modelling ofSwiss-model and compared with MabinlinB protein,revealing �46% sequence homology (http://swissmodel.expasy.org). The resultant homologue consisted of 55amino acids only (QPDFQRCCQQLRNISPPCRCPSL-RQAVQLTHQQQGQVGPQQVRQMYRVASNIPST),when compared to the original sequence of MO2.1 of 60residues. Hence, the remaining residues, QGPGR, wereincluded at the N-terminal end to get a homologuematching the sequence of MO2.1. This ‘model polypep-tide’ was used for all in silico analyses.

Protein–protein docking and dimer formation

Protein–protein interactions between the two monomerunits of MO2.1 were studied using the online serverCluspro (http://cluspro.bu.edu/login.php). Cluspro exe-cutes rigid body docking for two proteins to generatemillions of protein–protein complexes by varying relativedistances and orientations. A few thousand energeticallyfavourable structures were identified based on theprotein–protein interactions according to van der Waalselectrostatic and solvation free energies. The protein–protein complex structures were arranged based on theroot mean square deviation computed between the resi-dues of one monomer protein with that of another. Thedimer structures in which one monomer possesses ahigher number of residues compared with its neighbour

are considered to be representative for protein–proteincomplexes and many of them were arranged accordingto their interaction energies. The molecular simulationsand binding free-energy calculations were restricted onlyfor the lowest energy dimer, since this could be domi-nant and contribute largely to the dimer population.

Binding free-energy calculations (MM/PBSA and MM/GBSA)

Molecular dynamic simulations for the dimer solvated inwater as a solvent were performed using the Amber 11software (Case et al., 2010). The dimer configurationobtained from the protein–protein docking study was usedas an initial protein configuration for the MD simulations.The simulations were carried out in an isothermal–iso-baric ensemble with the simulation box containing a pro-tein dimer, water as solvent and ions to neutralize thesystem. Moreover, binding free-energy calculations wereperformed for 50 snapshots extracted at 50 ps intervalsfrom the MD trajectory of 2.5 ns. The binding free energyfor dimer formation is defined as follows:

DGbind ¼ Gcomplex � Gmonomer1 � Gmonomer2 ð1Þ

In the Molecular Mechanics (MM)-Poisson Boltzmannsurface area (PBSA), the free energy of each component isapproximated by separating the energies into molecularmechanics (electrostatic and van der Waals), polar andnonpolar solvation and entropic contribution:

G ¼ Emm � Gsolvation � TS ð2Þ

The molecular mechanics energy in the gas phaseEmm was computed with the same force field used inMD simulations:

Emm ¼ Ebond þ Eangle þ Etorsion þ Etorsion þ Eelec

þ EmdW ð3Þ

The solvation free energy Gsolvation was approximatedas the sum of polar and non-polar contributions:

Gsolvation ¼ GPB þ Gnonpolar ð4Þwhere the polar solvation term GPB was obtained byapplication of the Poisson–Boltzmann implicit solvationmodel (or Generalized Born in the case of GeneralizedBorn Surface Area [GBSA]) and the non-polar solvationterm Gnonpolar was approximated as linearly dependenton the solvent accessible surface area (SASA).

Results

Dimerization does not affect the coagulation activity

Protein profile analysis by polyacrylamide gel electro-phoresis showed the possible existence of monomer

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(treated with DTT; the presence of a �6.5 kDa band)and homodimer (untreated with DTT; the presence of a�14 kDa band) forms of MO2.1. As can be seen inFigure 1(A), the lane loaded with the E coli (inducedwith IPTG) sub-cellular proteins revealed the presence ofa homodimer, which was further reduced to the mono-meric form whose apparent molecular mass was found tobe comparable with that of MO2.1 in the crude seedextract.

Interestingly, all samples revealed similar coagulationactivities against synthetic turbid water with 35.6% activ-ity (Figure 1(B)), indicating no significant differencesbetween the recombinant monomer (85.0%) and homodi-

mer (83.1%) forms and the crude seed extract (85.0%).The identity of MO2.1 was confirmed by in-gel digestionof the corresponding protein bands and analysis by massspectrometry. The spectrum of a peptide identified froma �6.5 kDa band of the clone is shown in Figure 1(C).Having shown the possibility of dimerization, in silicoanalysis was performed to propose a theoretical modelfor the dimer.

Preliminary bioinformatics analysis of MO2.1

The protein sequence of MO2.1 verified by direct transla-tion from its 183 nucleotides (Broin et al. (2002),revealed a length of 60 amino acid residues (Figure 2

Figure 1. Protein profiles showing the homodimer and coagulation activity of MO2.1. (A) Electrophoretic analysis of a crude seedextract and E. coli sub-cellular fractions of a recombinant clone, showing MO2.1 as a �6.5 kDa monomer and a �14 kDa homodimer,respectively. Apparent molecular masses of the proteins were determined by comparison with low-molecular-weight range markersfrom Sigma (USA). (B) The percentages of turbidity reduction activity revealed no significant difference between the monomer ordimer forms compared with the crude MO seed extract. The figure represents the results of typical data from five independent assays,and the error bars were plotted by keeping a standard error of 5%. (C) Example of a collision–induced fragmentation product ionspectrum of a tryptic peptide from the monomer of recombinant MO2.1 protein. The detected a, b and y ions are shown as well as thecorresponding amino acid sequence (Mascot Ions Score: 135, e-value: 1.3e� 13).

Structure prediction of a flocculent protein 409

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(A)). A comparison of all the published proteinsequences of MO2.1 that were obtained from the UniProtand NCBI protein databases revealed differences at posi-tions 1, 13 and 23 (Figure 2(A)). The sequence of MO2.1

exhibits a hydrophobic ratio of 28% and contains Arg(13.1%), His (1.6%), Glu and Asp (1.6%), Cys (6.4%)and Gln (23.0%). The protein is highly positivelycharged with a theoretical pI of 12.6 showing a protein-

Figure 2. Bioinformatic analysis of MO2.1. (A) The nucleotide sequence of MO2.1 gene (183 bases) represents a sequence of 60amino acids. All the peptide sequences were obtained from the NCBI Protein database, and the differences are indicated by arrows.The investigated sequences are defined as follows: Gassenschemidt (1995): protein sequence from Gassenschemidt et al. (1995);Broin (2002): sequence mentioned by Broin et al. (2002); Suarez (2003): sequence mentioned by Suarez et al. (2003); GI1096142,GI998697, P24303: protein sequences obtained from the NCBI protein databases that were published based on the reference ofGassenschemidt (1995); GI127215, GI15626367, Q93YG0: protein sequences obtained from the NCBI protein database withreference to Broin et al. (2002); in this study, amino acid sequence used for the homology modelling. (B) Proteolytic cleavage mapof MO2.1: map showing various proteolytic cleavage points and the corresponding hydrolases for MO2.1, as generated by the ‘peptidecutter’ tool.

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binding potential of 2.81 kcal/mol, according to theBoman Index. Proteolytic analysis using ExPASy-peptidecutter disclosed the high susceptibility of MO2.1 againstseveral proteases, as mentioned in Figure 2(B).

Homology modelling based structure and electrostaticpotential of MO2.1

Earlier, Suarez et al. (2003) proposed a three-dimensional computational model based on the NMRstructure of napin (a homologous 2S-albumin) for theflocculent protein MO2.1, reporting a sequence identityof 71%. However, the sequence of this model peptidewas not presented. In order to improve the model, wehave performed a homology modelling study for thepolypeptide sequence of MO2.1 with MabinlinB protein(Figure 3(A)); the obtained homologous sequence con-sisted of 55 of the residues of MO2.1 (instead of 60).After completing the N terminal end with the remainingfive residues (QGPGR), an accurate model for MO2.1

was obtained. Furthermore, the conformational nature forthe added fragment was checked by computing theRamachandran angles associated with these residues.The (phi, psi) angles computed for these residues werearound (�60, �60), suggesting that the fragment had ahelical conformation. The final sequence revealed a100% sequence similarity with the sequence reported byBroin et al. (2002) and 97% similarity with the polypep-tide sequence of MO2.1 (accession P24303). In compari-son with the sequence #P24303 (reported based onGassenschmidt et al., 1995), the model peptide showed

amino acid differences at the 13th and 23rd positions.The proposed tertiary structure of the monomeric formof MO2.1 is presented in Figure 3(B), where the regionof five residues (QGPGR) that were not originallyobtained from homology modelling with MabinlinB arehighlighted in red. The model peptide had only threehelices and loop regions, while MabinlinB containedfour helical structures.

According to our in silico analysis, the first loopregion of MO2.1 was formed by Ile, Ser and Pro betweenthe 19th and 21st positions, while the second loop con-sisted of Gly, Gln, Val, Gly between the 40th to 43rdpositions. Having the secondary structure for MO2.1,electrostatic potential was computed as this would dictatethe interaction of the protein with various ligands or withother proteins. The electrostatic potential for the mono-mer (in water) was computed using an online server, pro-tein continuum electrostatics (http://curie.utmb.edu/area_man.html) and computed numerically by solvingthe Poisson–Boltzmann equation using a finite differencealgorithm (Figure 4(A) and (B)). The blue colour repre-sents positive domains corresponding to an electrostaticpotential of �3.0 kcal/mole (as such the protein has atotal charge of +6). However, the electrostatic potentialalso contours a few negative domains that are repre-sented in red. The cryptic behaviour of this positivelycharged protein interacting with positive ions can beexplained from the possible electrostatic interactionbetween the negative domains and positively chargedions.

Figure 3. Homology modelling of MO2.1. (A) Heterodimeric structure of MabinlinB: the homologous three helices are coloured incyan, while the blue colour represents the 4th helix. (B) Predicted structure for the MO2.1 monomer: the region coloured in redindicates the five amino acid residues (QGPGR) that completed the N-terminal end of the protein. The remaining cyan colouredhelices and loops were obtained from homology modelling.

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Protein–protein docking studies

To understand the dimer formation of MO2.1, a protein–protein docking study was performed with two mono-mers using the CLUSPRO software. As can be seen inFigure 5, the dimer has a compact structure and wasformed by both loop–loop and helix–helix contactsbetween the monomers. The three-dimensional structureof the dimer is shown in Figure 5(A) and its electrostaticpotential is depicted in Figure 5(B). The stabilization ofthe dimer due to residue–residue interactions is describedin the next section.

Binding free-energy calculations reveal stable dimeriza-tion of MO2.1

Binding free-energy calculations were performed usingthe MM_PBSA.PY module as implemented in Ambertools 1.5 (Case et al., 2010) for those configurations

obtained from MD simulations. The binding free ener-gies were calculated from both MM/PBSA and MM/GBSA models, which differ with respect to how theydescribe solvation free energy. In the former, the solva-tion free energy was computed from a numerical solutionof the Poisson–Boltzmann equation, while theGeneralized-Born equation was employed for the latter.The total free energies of dimer and monomeric forms ofMO2.1 are reported in Tables 1 and 2.

The difference in free energies between the dimerand monomer yields binding free energy that was foundto be responsible for the stabilization of dimer. Thecalculated values of the binding free energies are �86.7and �92.8 kcal/mol by GBSA and PBSA approaches,respectively, (Tables 1 and 2). Among the different indi-vidual contributions to the binding free energy, the vander Waals and solvation free energy could stabilize the

Figure 4. Electrostatic interactions of M.O2.1 according to the predicted model (A) and (B) are the two different views of thecomputed electrostatic potential for M.O2.1. The red colour indicates highly negative domains while blue indicates highly positivedomains of the protein.

Figure 5. Predicted tertiary structure and space-filling model of MO2.1. (A) Minimum energy structure of the MO2.1 dimer. The Glyof monomer 1 (40th residue) and Val (42nd residue) shown in cyan (ball and stick model) make contacts of a distance lower than4Å, whereas the residues making contacts within a distance range of 4–5Å are shown in yellow and orange. The blue and redcolours are used to distinguish the two monomers of the MO2.1 dimer. (B) Electrostatic potential of the predicted dimer. The redcolour indicates highly negative domains, while blue indicates highly positive domains of the protein.

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dimer formation, while the electrostatic interactionenergy destabilizes the formation of the dimer. Moreover,the contribution of polar solvation free energy to bindingfree energy was found to be many orders larger than thenon-polar counterpart. The reliability of computed bind-ing free energies was verified using the calculated stan-dard error. The relatively small value for the standarderror associated with binding free energy suggests thatthe time scale of MD simulations and statistics associ-ated with binding free-energy calculations are sufficientenough.

Discussion

The present study investigates the discrepancies in defin-ing the exact molecular mass of a coagulant protein fromMO through laboratory as well as in silico analyses. Theresults indicate the possible existence of monomer anddimer forms of MO2.1, which, however, both exhibit thesame coagulation activity. This observation is consistentwith earlier reports based on the use of homodimericfractions of MO2.1 that suggested that the flocculationactivity of MO2.1 is not affected by oligomeric changes(Katre, Suresh, Khan, & Gaikwad, 2008; Ndabigengesereet al., 1995; Prasuna, Chakradhar, Rao, & Gopal, 2009).It is worth mentioning the presence of diffuse bandsbetween 17 and 26 kDa under non-reducing conditions,

while up to 3 bands around 10 kDa are observed underreducing conditions only (García-Fayos et al., 2010).Such behaviour could be due to the formation ofoligomers with subunits that are probably linked throughS–S bonds. These observations motivated us toinvestigate the oligomerization of MO2.1 throughprotein–protein docking. There are limited theoreticalstudies pertaining to the structure and functionalities ofMO2.1 (Kwaambwa & Maikokera, 2008; Kwaambwa &Rennie, 2012; Suarez et al., 2003). In 2003, Suarez et al.proposed a 71% homologous structure for this proteinbased on the peptide sequence of napin, while the mod-elled polypeptide sequence was lacking. In order to pro-pose an accurate model for MO2.1 through stringenthomology modelling, we are proposing here a structureof MO2.1 with 100% sequence identity with the sequencereported by Broin et al. (2002). The sequence we haveused showed 97% homology with the #P24303 sequencepublished based on the original reference of Gassensch-midt et al. (1995).

In order to explain the ambiguities related to differ-ences in the molecular mass of MO2.1, the ability of thisprotein to form oligomers was investigated through aprotein–protein docking study. As shown in Figure 5, thedimer formation was favourable for MO2.1 because theprotein gains as much as 80–90 kcal/mole energy due tooligomerization. Moreover, the dimer formation was

Table 1. MM GBSA calculated binding free energy components of the dimer.

Contributions (kcal/mol)

Complex (dimer) Monomer (protein) Delta

Mean Std.E Mean Std.E Mean Std.E

VDW �859.75 1.89 �367.58 1.21 �124.57 .89ELE �4864.58 4.70 �2610.28 3.23 355.98 3.90G gas �5724.32 4.53 �2997.86 3.29 231.41 3.54EGB �2624.10 3.34 �1170.84 2.63 �300.43 3.46ESURF 61.32 .11 39.48 .08 �17.65 .06G sol �2580.78 3.32 �1131.35 2.61 �318.07 3.46G total �8305.10 3.04 �4109.22 2.29 �86.67 .61

Table 2. MM PBSA calculated binding free energy components of the dimer.

Contributions (kcal/mol)

Complex (dimer) Monomer (protein) Delta

Mean Std.E Mean Std.E Mean Std.E

VDW �859.75 1.89 �367.59 1.21 �124.57 .89ELE �4864.58 4.70 �2610.28 3.23 355.98 3.90G gas �5724.32 4.53 �2977.86 3.29 231.41 3.54EPB �2564.96 3.71 �1126.86 2.71 �311.24 3.65ECAVITY 42.49 .06 27.74 .05 �12.98 .04G sol �2522.47 3.70 �1099.13 2.61 �324.22 3.66G total �8246.79 3.30 �4076.99 2.51 �92.81 1.06

VDW – van der Walls energy; ELE – Electrostatic energy; G gas =VDW+ELE; EGB, EPB – Polar solvation energy; ESURF, ECAVITY – Non-polarsolvation energy.G sol = Polar solvation energy + non-polar solvation energy; G total =G gas +G sol.GB – Generalized Born; PB – Poisson–Boltzmann.

Structure prediction of a flocculent protein 413

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mostly stabilized by loop–loop contacts. In particular,the 40th residue of one monomer and the 42nd ofanother establish short distance contacts. The computeddistance between the centre of mass of these residueswas <4.0 Å, while other residue–residue contacts in thedimer possess a distance of 4 < r< 5.0. Cumulatively, thepair of residues involved in such contacts was found tobe 39:43, 59:60, 50:50, 35:56 and 35:46, which couldhave led to reduction in the SASA. Thus, solvent acces-sible areas of the residues in the monomer as well asdimer forms of the protein have been computed using anonline server GetArea (Fraczkiewicz & Braun, 1998).Further, the percentage decrease in SASA was computedusing the ‘naccess software’ to calculate SASAs of themonomer as well as dimer forms. The numbers of sur-face and buried atoms in the monomer are 339 and 134,while in the case of dimer they are found to be of 580and 366. Overall, the percentage of buried atoms in thecase of monomer was 28%, while it increased to 38% inthe dimer. Interestingly, the percentage of surface atomswas found to be 72% in the case of monomer, suggest-ing that the helices are not arranged compactly.

Functional activities of the monomer and dimerforms of MO2.1 showed no significant difference in thewater turbidity reduction activity (Figure 1(B)). Hence, itcan be concluded that the dimerization of MO2.1 doesnot interfere with its coagulation activity. As MO2.1 hasseveral proteolytic sites, the protein could form dimers toattain a stable state and retain its functionality againstharsh conditions. This hypothesis could be very wellobserved and supported in the case of the recombinantlyexpressed protein, as the protein apparent mass was oftenof �14 kDa. Further, this can be correlated with theresults of Broin et al. (2002) and Suarez et al. (2003),which showed an oligomeric expression of MO2.1 withmasses of �6 kDa and �10 kDa (Garcia-Fayos et al.,2010), respectively, under reducing conditions. In allcases, supplementing the lysis buffer with a reducingagent during the extraction and purification processescould avoid the oligomerization of the protein. Asrevealed by the protein–protein interaction studies, theformation of dimer and other oligomers are highlyfavourable. Thus, the molecular mass of the proteincould vary depending on the extraction protocols involv-ing different reducing and non-reducing agents, and dif-ferent buffer systems used in the extraction/purificationprocess. For example, Okuda et al. (2001) reported thatthe coagulant protein extracted by a salt solution exhibitsa molecular mass of 3 kDa, while the apparent massof the protein in a water extract was found to be of�12–14 kDa. This could be attributed to the electrostaticpotential and solvation free energies that play an activerole in dimer formation. The dominant contribution of

solvation free energy to binding free energy of the dimerexplains such an observation.

Though the experimental reports of MO2.1 proteinpertaining to its coagulation activity do not reveal thepossibility of oligomerization of MO2.1, our recent insilico studies on the interactions of monomer and dimerforms with trisodium citrate (TSC) evidences suchbehaviour (Okoli et al., 2012). The binding sites (Argresidues at positions 48 and 52) for TSC remain unal-tered in both cases and the binding strength in TSC wascomparable (Okoli et al., 2012). These results suggestthat the dimer formation does not alter its binding func-tionality even with small ligands like TSC and also sup-ports the indifferent coagulation activity observed in thisstudy using the monomeric and dimeric forms of theprotein MO2.1.

Conclusions

Based on the electrophoretic protein profile of the recom-binant protein, the MO coagulant protein (MO2.1) wasfound to exist as a dimer. It retains its functional activityas a coagulant, which was verified from turbidity reduc-tion experiments. Theoretically, structures for the mono-meric and dimeric forms of this protein were proposedby emphasizing the possibility of oligomerization.Homology modelling was used to predict the structure ofthe monomer, while protein–protein docking wasemployed for proposing the structure of the dimer. Theenergetics of dimer formation computed by its bindingfree energy (between �80 and �90 kcal/mole), showsthat the dimer formation is largely mediated throughloop–loop interactions. Though the molecular mass ofthe coagulant protein might be different, our results showthat such oligomerization does not result in any signifi-cant difference in the biological activity of the protein.Our work paves the way for future investigations on thestructure–function properties of coagulant proteins fromother vegetal species in relation to their oligomerizationand biological activity.

Acknowledgements

ARP acknowledges European Commission’s Erasmus MundusExternal Cooperation Window (EURINDIA) and the support ofMrs. Alphonsa Lourdudoss, Coordinator at the Royal Instituteof Technology (KTH), towards his postdoctoral scholarship.

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