Structural and Functional Interferences from a Molecular Structural Model of Xenocin Toxin from Xenorhabdus nematophila

Bacteriocins constitute the most abundant and diverse family of microbial defense systems. They are produced and secreted to inhib it the growth of closely related, competing bacterial species inhabiting a common ecological niche. One such bacteriocin designated as xenocin, is produced by entomopathogenic bacterium Xenorhabdus nematophila. In our earlier study, we have shown its regulation under SOS conditions and its activity against wide range of bacteria isolated from the gut of insect. In this study three dimensional structure of xenocin has been deciphered with the assistance of automated homology modelling and verified by VERIFY-3D program as well as Ramachandran plot 2.0. Three domain organisation; Translocation (T), Receptor (R) and Catalytic domain (C) has been observed and their structures were studied by CHIMERA software (UCSF). Protein disorder structure and Average Area Buried Upon folding (AABUF) in first 100 amino acid residues of Translocation domain is determined by Globplot and ProtScale software respectively. Conserved amino acid residues in the putative active site of the catalytic domain of xenocin have been deciphered with mult iple sequence alignment.


Introduction
Bacteriocins are ribosomally encoded toxins produced by bacteria to inhibit the growth of closely related bacterial strain(s) [1]. They are produced by almost all the major lineages of Eubacteria and Archebacteria and are structurally, functionally, eco logically diverse [2]. Bacteriocins are generally produced in the cytoplasm and secreted in the extracellular mediu m by the producer where they target specific receptors on the surface of susceptible cells. Induction of to xicity in target cells occurs by different mechanisms including enzy mat ic nuclease (DNase or RNase) as well as pore formation in the cytoplasmic memb rane [3]. Their structure comprises of three distinct domains organization: (i) a domain involved in recognition of specific receptor, (ii) a domain involved in translocation, and (iii) a do main responsible for their to xic activ ity, with average molecular mass of ~25 to 80 kDa [4].
Xenorhabdus nematophila is a motile gram-negative entomopathogenic bacteriu m belonging to the family Enterobacteriaceae [5]. It forms symbiot ic association in the gut of a s o il n e mato d e o f fa mily S tein ern ema tid a e [ 6 ]. X. nematophila is known to secrete several extracellular products, which include lipase(s), phospholipase (s), protease(s), and several broad spectrum antibiotics [7,8] when grown under standard laboratory conditions. These products are believed to be secreted in the insect hemoly mph during the stationary phase of bacterial growth. The degradative enzy mes are responsible for the breakdown of macro molecules of the insect cadaver to provide nutrient to the developing nematode, wh ile the antibiotics play a major role in the suppression of contamination of the cadaver by other soil microorganis ms. In our earlier study, we have isolated and characterized xenocin operon encoded by the genome of X. nematophila. Results showed that the transcription of xenocin was upregulated by iron depleted condition, high temperature and in the presence of mito mycin C. Reco mb inant xenocin-immunty protein comp lex showed broad range of antibacterial effect, not only limited to the laboratory strains but also to the bacteria isolated fro m the gut of H. armigera [9]. Therefore, it is essential to study the structure of such an important antibacterial protein in detail. In this study, similar proteins fro m other sources has been identified with protein-protein blast and phylogenetically analy zed. Three dimensional structiure of xenocin is deciphered by automated homology modeling. Protein d isorder and average buried area upon folding of first 100 amino acid residue fro m the Translocation domain o f xenocin is analyzed by Globplot and ProtScale fro m Expasy. Conserved amino acid residues M odel of Xenocin Toxin from Xenorhabdus nematophila in the putative active site of catalytic domain o f xenocin have been predicted by multiple sequence alignment and surface viewed with CHIM ERA software.

Sequence Alignment
The protein data banks were searched against xenocin protein, using BLA STP (http://www.ncbi.n lm.n ih.gov) program. A ll protein sequences were obtained fro m NCBI sequence database (http://www.ncbi.n lm.n ih.gov)

Phylogenetic Analysis
All sequences that matched with xenocin were aligned by CLUSTA LW (Mac vector 7.0), and a tree was constructed using neighbour-joining method, with the best tree mode in the Mac vector version 7.0 (Oxford Mo lecular, England) program.

Molecular Homolog y Modeling
Three dimensional structure of xenocin was produced by using ESyPred3D (http://fundp.ac.be/urbm/bioinfo/esypred/) automated homology modeling server. Xenocin shares ~30% sequence identity with E3 co licin and as such the three dimensional structure of the latter determined by X-ray crystallography [10] was used as a specified template for homo logy modeling of the former in automated ho mology modeling. Predicted structure was verified by VERFIFY-3D (http://nihserver.mbi.ucla.edu/ Verify_3D) and further analysed by Ramachandran plot 2.0 software (http://dicsoft 1.physics.iisc.ernet.in/rp/select.html)

Prediction of Protein Disorder and of the Average Area B uried Upon Fol ding
Protein d isorder was determine by Globplot (http:// globplot.emb l.de) and Prediction of the average area buried upon folding (AABUF) was calculated with the ExPASy tool ProtScale (http://us.expasy.org/tools/protscale.html)

Acti ve site Anal ysis
After the final model was built, the conserved amino acid residues in putative active site of catalytic do main of xenocin was explored mu ltip le align ment and surface viewed with CHIM ERA software (http://www.cgl.ucsf.edu/chimera)

Phylogenetic Analysis
Phylogenetic analysis of the xenocin was done by preparing phylogenetic tree, using neighbor-joining method, with the best tree mode in the Mac vector version 7.0 (Oxford Molecular, England) program as shown in Figure 1.
It showed that xenocin fro m X. nematophila formed a separate branch fro m other bacteria in the very beginning and was located at opposite position with reference to toxin fro m Klebsiella oxytoca. To xin fro m Klebsiella pneumonia and E. coli cloacin DF13 form a d istinct cluster where as colicin like E6 and E3 fro m E. coli formed their own separate cluster. Protein-protein b last showed that xenocin is just 31% similar to cloacin of K. pneumonia which is located at distal position fro m xenocin in phylogenetic tree however, whereas it is only 30% similar to colicin E3 wh ich is located proximal to it phylogenetically.

Molecular homolog y modeling and predicti on of average area buried
Protein-protein blast of xenocin protein showed 30% similarity with E3 and E6 colicins. Moreover, crystal structure of E3 colicin has been resolved [10]. Therefore, three dimensional structure of xenocin was predicted by EsyPreDSD using E3 as temp late. Predicted structure by homology modeling consisted of a stem corresponding to receptor domain R and two g lobular heads of translocation domain T and catalytic do main C respectively as shown in Figure 2 (a). Analysis usingVerify3D programme showed more than 94% of the positive score value and 80% h igher than 0.2. Ramachandran plot calculation showed 96.44% of the residues in the favoured and allowed regions Figure 2 (b). This analysis indicates that the model has a good quality.
There are three distinct steps in the killing of the sensitive cells by colicin, each of which is carried by separate domain. The first step is receptor binding to the surface of the target cell which is mediated by the receptor do main R, wh ich lies in the central part of the primary sequence. The N' terminal translocation domain mediates the second step, translocation fro m the outer memb rane receptor to the colicin's targets in the cell. Finally, the killing activity resides in the carbo xyterminal C do main. Since, xenocin having three do main organizations; Translocation (T), Receptor (R) and Catalyt ic domain (C) each could have their own distinct functions.  In colicin E3, receptor do main plays a majo r role in the first step, that is the interaction with target cell. BtuB is outer memb rane protein targeted by E3 colicin. Binding of E3 with BtuB p rovide a scaffold for protein co mplex to sit on the surface of the target cells. Moreover, this interaction provides signal required for the conformational change in E3 colicin for the removal of immunity fro m the E3-immunity protein complex. N' terminal of BtuB p rotein form a floor of a pocket that is about 25 Å deep and 25 Å in diameter. This floor is entirely made up of hydrophobic residues. The exposed hydrophobic residues of the R domain may anchor it to the floor of BtuB binding pocket [10]. In xenocin receptor domain R consists of antiparallel helical hairp in that extends fro m 328 to 476 amino acids as shown in Figure 3 (A) with first helical arm of this coiled co il formed by residues 328 to 407. A hairpin structure is formed by residues 408 to 413. Second helical arm extends fro m residues 414 to 476. In xenocin receptor domain, the upper helical reg ion is mo re hydrophilic where as the lower reg ion including the hairpin structure is mainly co mposed of hydrophobic amino acids as shown in Figure 3 (B and C). Most of the hydrophobic residues in these regions are exposed to the surface which may play crit ical and important role in interacting with receptor on the target cell.

Translocation Do ma in
Translocation domain in co licin is usually highly disordered in nature and this attribute helps them to interact with other outer membrane proteins. Bacteriocins are broadly classified into two groups, groups A and B, based on cross-resistance. Group A co mprises of bacteriocins that are translocated by the Tol system, such as colicins A, E1 to E9, K, L, N, S4, U, and Y, while group B co mprises of bacteriocins that use the TonB system, such as colicins B, D, H, Ia, Ib, M, 5, and 10 [3]. N' terminal of Tol dependent colicin contain sequence recognition motif DGSGW for the binding with TolB, a periplasmic protein involved in translocation fro m outer membrane to cytoplasmic memb rane. Xenocin primary sequence from 1 to 327, forms N' terminal translocation domain T. Ho wever, only 52-327 amino acid residues forms a jelly ro ll surface composed mainly of β sheets and α helical structures as shown in Figure 4. N' terminal xenocin is glycine rich, it consists of 17 % glycine in first 51 amino acid residues whose structure was not shown by homology modeling. Though, xenocin has maximu m similarity to colicin E3 it lacks the tol B recognition motif DGSGW p resent in the latter fro m amino acids 25-39. Instead, xenocin sequence contain a recognition motif EAMAI fro m amino acids 55-59, having homology to tonB box b inding motif [11]. This indicates that xenocin may have different mode of translocation as compared to colicin E3.
The protein disorder in first 100 amino acid residues is deciphered by Globplot (http://globplot.embl.de) as shown M odel of Xenocin Toxin from Xenorhabdus nematophila in Figure 5 (A) and average area buried upon folding predicted with the ExPASy tool ProtScale shown in figure 5 (B). Results showed that maximu m disorderedness was observed in the first 50 residues as shown with blue bar in figure 5 (A) which corroborate with our three dimensional structure which does not showed any structure of this reg ion. This could be due to the presence of 17 % glycine in this specific region and contribute no electron density as observed earlier in the case of E3 colicin, where glycine content was 47% in this region [10].
Region between 55 to 65 residue have most ordered structure as predicted by ProtScale shown in figure 5 (B), and interestingly this contain the sequence recognition motif EAMAI in the residues 55-59 for b inding with to a periplasmic protein Ton B.

Catalytic Do main and Active Site Prediction
Inner memb rane peptidases are important for the colicin cleavage which is necessary for killing the target cells [12]. Generally, only the catalytic domain which is resistant to proteases of inner membrane translocate to cytoplasm due to its amphiphilc nature [13]. Once they enter the cytoplasm they show detrimental effects on the target cells by targeting either DNA or RNA which ultimately inhib it the cell growth.  Third domain of xenocin is catalytic domain C located at C terminal, consisting of five straight anti parallel h ighly twisted β sheets and three α helixes at its N terminal as shown in Figure 6. Catalytic do main of xenocin possessed RNase activity as showed in in vitro RNase assay [9]. In the catalytic domain of xenocin, arg inine contributes 7.5% whereas lysine contributes 13.2%. In totality, they provide net positive charge to catalytic do main. It might be possible that positive charge on the basic amino acids help in binding by neutralizing the negatively charged phosphate group of the substrate RNA backbone. The general catalytic mechanism by wh ich RNA has been hydrolyzed has been thoroughly studied by protein engineering and crystallography [14]. RNase A, has two active His residues that cooperate during the catalytic cycle. One act as a general base and extract a proton fro m the ribose 2'-OH, thereby catalyzing the nucleophilic attack of this hydroxy l g roup on the 3'-phosphate group, leading to cyclic intermed iate, wh ile other serve as catalytic acid during the first cyclizat ion step. Their catalytic roles are reversed during the subsequent hydrolysis of the cyclic intermediate. Other ribonuclease, such as barnase and colicin E3, proceed probably through the similar mechanis m but His and a Glu function as catalytic residues [15]. Catalytic domains fro m all the protein sequences that matched with xenocin were aligned by CLUSTA LW (Mac vector 7.0). Results showed that D535, H538, E542, R570, H551 and K564 are highly conserved and may form active site for catalytic domain. Interestingly, in predicted three d imensional structure of catalytic do main as shown in Figure 7 the presence of two His residues (H538 and H551) on the surface might be responsible to hydroly ze RNA by similar mechanis m as followed by RNase A. Fro m the structural analysis of the catalytic domain, localization of aspartic acid D535 on the surface and nearest to H538 might be responsible to hydrolyze RNA by similar mechanism as followed by barnase. Spatial location of R570 and K564 on the surface as well as in the vicin ity of D535, H538, E542 and H551 may help in b inding by neutralizing the negative charged RNA substrate due to the phosphate group.

Conclusions
The antibiotic proteins are named after the bacterial species that produce them; e.g., the "colicins" are derived fro m Escherichia coli [16], the "pyocins" are derived fro m Pseudomonas aeruginosa [17], "klebocin" is derived fro m Klebsiella pneumonia [18] and "xenocin" is derived fro m X. nematophila [9]. Most bacteriocins have a common mo lecular o rganizat ion consisting of three functional domains, the receptor binding, membrane t ranslocation, and cytotoxic domains. In gram-negative bacteria, the lethal action of bacteriocins involves the following essential steps. After release fro m the host cell, the bacteriocin b inds to the specific surface receptors on the target cells, such as vitamin B12 receptor Btu B o r iron siderophore receptor FepA 19 through their Receptor do main [4]. In xenocin 328-476 forms a putative receptor which may bind to the protein present on the surface of target cells. This binding is followed by import into the cells with the help of either Ton proteins (Exb B, Exb D, and TonB) or Tol proteins (To lA, -B, -Q, and -R) [2,20] for interaction with the target molecule. In our case 55-59 residues of xenocin form recognition motif specific for TonB protein, this may direct its entry into the cytoplasm by Ton pathway. Bacteriocins use variety of strategies to induce cell death through their cytotoxic domains. The pore-forming bacteriocins create voltage-gated channels, causing depolarizat ion of the cytoplasmic membrane [21,22], while nucleases cleave tRNA or rRNA at specific sites to inh ibit protein synthesis [23,10] o r degrade nucleic acids nonspecifically in the target cells [24,25] In our earlier study with xenocin we have shown invitro RNase activity where as no DNAase activity had been observed. Therefore, we presume that after processing in the periplas m, only catalytic do main enters the cytoplasm of target cells and perform its detrimental effect. Conserved amino acid residues such as D535, H538, E542, R570, H551 and K564 o f the catalytic domain which may involve in such activity were determined by multip le sequence align ment. Moreover, their presence on the surface to interact with negatively charged substrate is also deciphered by the surface view of all these amino acid residues. Validation of predicted amino acid residues in the putative active site of catalytic do main of xenocin, is going on with site directed mutagenesis experiments in our laboratory.