Introduction

Clostridium botulinum is a spore-forming anaerobic bacterium causing avian and mammalian botulism outbreaks. Physiological group-III strains of C. botulinum are capable of producing C2 and C3 toxins, in addition to botulinum neurotoxin serotypes C and D (Moriishi et al. 1991, 1993). C2 and C3 toxins are belonging to ADP-ribosylation superfamily or bacterial ADP-ribosyltransferases (BADPRTs), which is categorized into four types designated I–IV (Holbourn et al. 2005). Type I consists of heat-labile enterotoxin A, cholera toxin A, and pertussis toxin S. Type II includes diphtheria toxin A and exotoxin A and Type III encompasses C3-like exotoxins. Type IV contains C. botulinum C2 toxin, C. perfringens ι-toxin, C. difficile Cdt toxin, C. spiroforme toxin, and Bacillus cereus vegetative insecticidal protein 2 (VIP2). Type IV is commonly known as binary toxin A (BTA) that transfers an ADP ribose moiety of NAD+ to G-actin and F-actin (Tsuge et al. 2008; Wiegers et al. 1991).

C2 toxin consists of an enzymatic component C2I and a binding/translocation component C2II (Ohishi et al. 1980). C2I composes of an adapter domain and a catalytic domain related with VIP2 and iota toxin a (Ia) (Aktories and Barth 2004; Tsuge et al. 2003). In C2 toxin, a functional motif serine–threonine–serine sequence (STS), a α1-helical structure and folding of a core β-strands surrounded by α-helices are highly conserved to maintain its structure–function link similar to other BTA family members (Takada et al. 1995). Glu187 is a catalytic residue in C2I that deprotonates Arg177 in G-actin. Glu189 forms H-bond with the O′2 on nicotinamide ribose that stabilizes the oxocarbenium cation, leading to α-selective ADP-ribosylation (Barth et al. 1998; Domenighini et al. 1994; Domenighini and Rappuoli 1996; Fujii et al. 1996; Jank and Aktories 2013). C2II composes of five domains (C2II20, D1–D4) in which D1–D3 consist of C2I binding components evolutionarily related to protective antigen (PA) of anthrax toxin and iota toxin b (Ib) (Barth et al. 2004; Leppla 1995; Schleberger et al. 2006). D4 domain structure has no similarity with D4 of PA and Ib (Blöcker et al. 2000). D4 contributes to stabilize a conformation needed for receptor binding and cytotoxicity (Blöcker et al. 2000; Varughese et al. 1999). Glu307 is the only negatively charged amino acid sharing its function of pore-forming and C2I–C2IIa complex formation, which phylogenetically close with PA (Blöcker et al. 2003a, b; Benson et al. 1998; Nagahama et al. 2003; Petosa et al. 1997).

After proteolytic cleavage, a residual fragment C2IIa released from C2II binds with C2I and then recognizes on the cell surface receptors (Aktories and Barth 2004; Blöcker et al. 2000; Eckhardt et al. 2000). The uptake of C2 toxin is mediated by dynamin-dependent pathways and endocytosed by processes dependent on clathrin and RhoA (Pust et al. 2010). C2IIa heptamer is inserted into the membrane and forms pores in a late endosome under acidic pH and then bound C2I is translocated into the cytosol (Blöcker et al. 2003a, b). C2I catalyzes the ADP-ribosylation at the residue Arg177 of G-actin and exhibits to reduce the ability of actin to undergo polymerization, leading to disruption of the cytoskeleton architecture (Sterthoff et al. 2010; Tsuge et al. 2008).

Site-mutagenesis studies evidenced its substrate specificity, recognition of NAD-binding core, and channel/pore-forming property (Barth et al. 1998; Blöcker et al. 2003a, b; Fujii et al. 1996; Han and Tainer 2002; Lang et al. 2008; Schleberger et al. 2006). Conserved residues contribute to the overall stability of a protein structure and function (Kowarsch et al. 2010; Vitkup et al. 2003). Evolutionary constraints on a disease-related gene are relaxed allowing it to accumulate mutation after gene duplication. Physical and evolutionary restraints also signify the associations between patterns of amino acid replacement and protein structure (Han et al. 1999). Such constrictions reflect on the substitution rate of residues in different secondary structural environments and of different solvent accessibilities (Tseng and Liang 2006). Thus, evolutionary forces determine its structure–function integrity, avirulence state and disease-susceptibility (Chellapandi et al. 2013; Chellapandi 2014; Prathiviraj et al. 2015; Xia and Levitt 2002).

Enzymatic inactive mutants from C2 toxin (Fahrer et al. 2010a, b) and gene-mediated adjuvants from Escherchia coli heat-labile toxin (LTB) (Cunha et al. 2014; Gil et al. 2013; Krüger et al. 2013) and non-catalytic cholera toxin A (Wan et al. 2014) were recently developed for therapeutic applications. No vaccine has been designed or developed for C2 toxin-mediated diseases in poultry and veterinary animals to date. Thus, the present study was aimed to reveal how evolutionary forces enforcing on BTA family are useful measures to determine the functional diversity and avirulence states of C2 toxin. Finding structural constraints in C2 toxin would further improve the accuracy and reliability of our screening and selection of its avirulent toxins with increased immunogenic nature.

Materials and Methods

Analysis of Phylogenetic Diversity

Crystallographic structural information for C2I (PDB ID: 2J3V; UniProt: 069275) and C2II (PDB ID: 2J42; UniProt: 086171) were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) and corresponding protein sequences retrieved from the UniProt database (http://www.uniprot.org/). The similarity hits for these template sequences were searched from NCBI-non-redundant protein database by BlastP program (Altschul et al. 1997). Multiple sequence alignment was conducted with ClustalX 2.0.11 software (Thompson et al. 1997). Estimates of phylogeny for sequence alignments were obtained using the neighbor joining algorithm in the MEGA 5.05 software (Tamura et al. 2011) with 1000 bootstrapped alignments. The constructed trees were visualized and edited with MEGA.

Analysis of Functional Divergence

The type I (θ I) and type II (θ II) functional divergence coefficients along with the rate for gamma distributions (α) were examined across the BTA family by DIVERGE 1.04 (Gu and Vander Velden 2002). Phylogenetic functional divergence and its parameters of this family were analyzed by SplitsTree 4.0 software using BioNJ algorithm (Huson and Bryant 2006). Recombination events and mutation rate were detected by RDP 3.0 software using Recomb2007 algorithm (Martin et al. 2010). Hierarchical network parameter for detecting recombination events was computed by CYTOSCAPE 3.0.1 software (Shannon et al. 2003).

Genetic Diversity and Darwinian Selection

Evolutionary genetic analyses (R, I, d, S, π, D) were performed with programs in the MEGA. Gene and amino acid substitution patterns across this family were calculated by the maximum composite likelihood method. The purpose of Tajima’s test was to identify sequences which do not fit the neutral theory model of equilibrium between mutation and genetic drift (Tajima 1989). Sites subject to Darwinian selection and evolutionary rates were calculated by a selective strength computed from Ka/Ks ratio and dN/dS ratio (ω). The Ka/Ks ratio was calculated with Ka/Ks calculator from different evolutionary models (Zhang et al. 2006). Codon-based likelihood in synonymous and non-synonymous substitutions was used to estimate dN/dS ratios (ω) using HyPhy 1.0 program (Pond et al. 2005).

Estimation of Evolutionary Rate

A similarity scoring matrix calculated from estimated values was detected a protein homolog which was evolutionarily related to C2 toxin as described earlier (Lio and Goldman 1999; Pearson 1998). Global alignment of two protein sequences was carried out by EMBOSS ALIGN tool using different PAM matrices with the Needleman–Wunsch algorithm (http://www.ebi.ac.uk/Tools/psa/). Evolution rate was estimated between C2 toxin and its homologs based on evolutionary relatedness and sequence similarity between amino acids as reflected in substitutions matrixes (Udaya Prakash et al. 2010).

Identification of Mutable Residues

The crucial residues involving in structural and functional stability of this toxin were identified from the literature. Conserved motifs in the sequences were identified from the NCBI conserved domain database (Marchler-Bauer et al. 2015). The mutable residues that bring changes in the function of C2 toxin were predicted from its structure by HotSpot Wizard (Pavelka et al. 2009). Rate4Site 2.01 program was employed to estimate the evolution rate of detecting mutable residues from a probability of its multiple sequence alignment (Mayrose et al. 2004).

Analysis of Coevolution Sites in Local Structural Environment

Secondary structural elements of this toxin and its features including solvent accessibility and H-bonding were predicted with JOY server (Mizuguchi et al. 1998). A coevolution (coupled mutation) site on the local structural environment was inferred from its sequence by a CMAT program with profile-based adjustment and mutual information (Jeong and Kim 2012). Network parameter was calculated for detecting coevolved pairs by CYTOSCAPE 3.0.1.

Analysis of Structural Stability upon Point Mutation

CUPSAT was used to predict the mutant residues that stabilize its structure upon point mutation and to assess the amino acid environment of the mutation site based on amino acid-atom potentials (Parthiban et al. 2006). Structural stability and virulence state of resulted mutants were predicted with SDM server using a statistical potential energy function (Worth et al. 2011). The structural stability score was calculated from the energy function by using environment-specific aminoacid substitution frequencies within homologous protein families.

Analysis of Structural Constraints in Avirulent Toxins

The protein folding rate of all structural classes in C2 toxin and its avirulent mutants was predicted from the sequences by FoldRate program using multiple regression technique (Gromiha et al. 2006). Avirulent mutants with more structural stability were chosen by capturing near-native ensembles of their backbone conformations using Backrub Ensemble (Smith and Kortemme 2008). Structural flexibility in avirulent toxins was analyzed by PredyFlexy server using a structural alphabet and combining classical X-ray B-factor data (De Brevern et al. 2012).

Analysis of Immunogenic Nature of Avirulent Toxins

Using the potential hydrophilic regions, immunogenic nature (antibody epitopes) of avirulent toxins was predicted from their amino acid sequences. The hydrophilicity, flexibility, accessibility, turns, exposed surface, polarity and antigenic propensity of polypeptide chains were selected as parameters to predict the location of continuous antibody epitopes. Antibody epitope prediction tool in the Immune Epitope Database (http://tools.immuneepitope.org/bcell/) was used to predict the sites that produce an immunogenic response against C2 toxin and its avirulent toxins, according to a semi-empirical method described by Kolaskar and Tongaonkar (1990) (Kolaskar and Tongaonkar 1990).

Results

Molecular Phylogeny of C2 Across the BTA Family

A dataset for the BADPRTs superfamily consists of 28 complete sequences, related to C2 toxin, obtained from different family members (Supplementary Fig. S1). C2I and C3 are related together and shared their phylogenetic resemblance with Ia and pertussis toxin S5. A phylogenetic proximity is found between C2II and LTB. Cholix toxin is a unique protein and corresponds with C3-like exotoxins. A dataset for inferring phylogenetic relationship of BTA family comprises 35 sequences whose crystallographic structures are to be solved to date (Fig. 1). C2I, C2II, and C3 are clustered separately in the phylogenetic tree in which C2I is evolutionarily related to Ia and VIP2 whereas C2II is similar to Ib. This suggested that both components of C2 and iota toxins share a common evolutionary origin. In contrast, C3 toxin shows a phylogenetic closeness to Toxin2A and EDIN (Epidermal cell differentiation inhibitor) of this family, but not related to C2 toxin as such.

Fig. 1
figure 1

Phylogenetic tree of Clostridium botulinum C2 toxin reconstructed from the similarity sequences of available crystallographic structures. Branch lengths are proportional to evolutionary distances. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed

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Functional Divergence of C2 Across the BTA Family

Our results described that all sites evolve at a roughly same rate across C2II/Toxin2A cluster and many sites evolve slowly, but a few evolve rapidly among clusters (Table 1). Apart from a cluster of C2II/Toxin2A, a strong rate of heterogeneity among sites is detected across other homologous clusters. Type I functional divergence after gene duplication results in altered functional constraints with different evolutionary rate between duplicate genes of C2II/Toxin2A cluster and C2I/Ia cluster. No altered functional constraints, but radical change in amino acid property is detected between duplicated genes of C2II/C3 and VIP2/C3 clusters that may be arising due to type II functional divergence. Neutral selection is imposed on the functional divergence across the clusters of C2II/VIP2, C2I/C3, VIP2/Toxin2A, and Ia/C3.

Table 1 Coefficients of functional divergence between homologous clusters of binary toxin A family, estimated by diverge
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Recombination Rate of C2 Across the BTA Family

Evolutionary preference for recombination robustness is much stronger than mutational robustness (Supplementary Table S1; Fig. S2). Recombination analysis of our study predicted two major recombination networks resulting across this family, where the C2II gene in C. botulinum type C may be acquired from the C2II gene of D phage through C3I gene of C phage by horizontal gene transfer event. A gene encoding Bcer toxin from B. cereus has served as a major parent to combine many segregation sites in directing the recombination process in the evolution of C2, C3, C3stau2, Ia, and EDIN. It clearly implied that C2 and C3 toxins might be evolved separately for their unique functions from those phages or bacterial origins at different evolution rates via a short recombination path.

Evolution Rate of C2 Across the BTA Family

We have employed different substitution matrices (10–500) to calculate its evolution rate from homologs as shown in Fig. 2. As a result of substitution score, a strong sequence conservation is observed between C2I/C2II; C2II/Ib; C3I/C3II. It reflected that evolution rate and acceptable substitution mutations are lower than as anticipated in other toxins. The highly acceptable mutations are also detected between C2I/Ia; C2II/VIP2; C3II/Toxin2A. Substitution rate and sequence conservation are predicted to be low between C2I/C3I; C2II/C3II; C2II/Toxin2A. Moreover, C2 shares its residue substitutions within the iota toxin, whereas residues in C3 substitute with VIP2 and Toxin2A.

Fig. 2
figure 2

Amino acid substitution patterns for comparison of Clostridium botulinum C2 toxin with C3 toxin and binary toxin A family

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Genetic Diversity of C2 Across the BTA Family

A site-specific rate change is related to functional divergence during protein evolution. Genetic diversity analysis shows that gene (π 0.479) and protein (π 0.452) sequences are diverged within the BTA family at a slow recombination/mutability rate (Table 2). Phylogenetic distance for gene diversity (d 3.254; R 1.11) is higher than protein diversity (d 2.929). Darwinian positive selection acting on gene function (D 4.3) is rather than on protein function (D 3.3). The synonymous substitution rate of C2 is higher than non-synonymous substitution rate to select and purify its function from BTA family. However, the selective strength (Ka/Ks; Ω) supports the fitness of the C2 function as such by neutral evolution.

Table 2 Estimates of genetic diversity and Darwinian selection for binary toxin A family
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Coevolution on Local Structural Environment of C2

Coevolution is significantly acted on the residues (64%) in helices of the secondary structure of C2I, which in turn, to change coevolved pairs as β-strands (Fig. 3). About 15% changes is accounted between residues in helices and residues in coils due to coupled residue mutations. No major adjustments in C2 are observed in other structural environment at secondary level. Coevolution sites (65%) are majorly accounted in residues involving in the solvent accessible surface area (SASA). Conversely, the local structural environment may be altered from buried surface area to SASA and vice versa (30%). About 38% of the residues is not contributed in the H-bonding. H-bonding (24%) and non-hydrogen bonding (25%) residues destabilize H-bonding frequency in coevoled pairs. Coevolution on H-bonding residues radically rearranges the H-bonding pattern with non-H-bonding pairs. As a result, the order of primary coevolution sites is detected as 393 > 348 > 39 > 29 > 16 > 73 from the coevolution network with shortest path of 4%.

Fig. 3
figure 3

Calculation of secondary structural types (a), solvent accessibility (b), H-bonds (c), and H-bonding frequency (d) for coevolved sites identified in the C2I structure. Within the coevolved site pairs, if both residues are buried or accessible, they are shown as ‘BUR–BUR’ or ‘ACC–ACC’, respectively. ‘HBDY–HBDY’ and ‘HBDX–HBDX’ indicate cases where both coevolved residues are involved or not involved in H-bonding correspondingly. ‘HBDX–HBDY/HBDY–HBDX’ indicates cases where at least one residue is involved in H-bonding. Values in the parenthesis show mean and standard error of estimated from the distribution of structural property values for randomly selected non-coevolved residue pairs. H α-Helix, B β-strand, C coil

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Screening and Selection of Avirulent Toxins

Conserved domain analysis indicated that C2I has two functional motif regions such as [Y252]–[R265]–[R299]–[S348T349S350]–[S361]–[E389]–[F398] and [F384]–[E387]–[E389]. Arg is identified as a site for conformational flexibility of the ligand binding pocket. Evolution rate is calculated to be low for the residues selected for point mutation, suggested that these residues contribute a crucial role in its molecular function (Supplementary Fig. S3). We have screened 29 mutants from wild-type C2I and 21 mutants from C2II, which are detailed in Supplementary Tables S2, S3. Only 17 mutants from wild-type C2I and 16 mutants from C2II were selected as avirulent mutants with no radical changes in their secondary structural elements, but overall native-like structures are destabilized by point mutations. Finally, four C2I and five C2II avirulent mutants with more structural stability (−ΔΔG) were selected for further evaluation (Table 3). It shows that the exchange of the STS motif in C2I causes a decrease in structural stability compared to E389K and F398Y mutants (ΔΔG −1.94 to −1.65 kcal/mol). Channel/pore-forming property of C2II is substantially changed in its mutants. F456S and F456A have shown more structural stability (ΔΔG −1.96 to −1.79 kcal/mol) without change in H-bonding patterns. Solvent accessibility of C2I and C2II avirulent mutants is drastically reduced as low as C2 toxin. The local structural environment in C2I avirulent mutants is stabilized by unsaturated H-bonding resulted from saturated H-bonding, but H-bonding pattern in C2II mutants is not destabilized as such.

Table 3 Amino acid mutations that stabilize the overall structure of C2 avirulent toxins
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Protein Fold Rate of Avirulent Toxins

The folding rate on structural classes, particularly a mixed class (α + β and α/β), of C2I, is decisively changed the folding process of its avirulent mutants (Fig. 4). The fast fold rate is detected in the mixed classes of T349H, E389K, and F398Y compared with C2I. The minor and major changes in fold rate are detected in all-β class and all-α class of C2I mutants, respectively. The fast fold rate is resulted in all-α class, which was higher than all-β class of its mutants. C2II mutants not including E399C and F456A accomplish the fast fold rate in many structural classes, and no major changes detected in the fold rate on all-α class. The fold rate on all structural classes of C2II is faintly lower than F456S and F456H.

Fig. 4
figure 4

Fold rate prediction for comparison of structural classes in C2 toxin and its avirulent mutants

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Backbone Conformation of Avirulent Toxins

We generated different conformational ensembles of the C2 toxin carrying a single amino acid mutation, which are shifted from native proteins (Supplementary Fig. 4). The best backbone ensembles are captured for S350R (−413 kcal/mol) and F398Y (−411 kcal/mol) from C2I structure. E389K (−405 kcal/mol) and T349V (−400 kcal/mol) also yield the stable ensembles with insignificant conformational flexibility. Similar to C2I mutants, we obtained stable backbone conformations for F456H (1083), F456A (1089), and E399C (1064) from C2II structure (1076 kcal/mol). F456S (587 kcal/mol) has not shown significant near-native shifts similar to the C2II structure due to localized structural flexibility. It is suggested that C2II mutants apart from F456S are able to reduce the channel/pore-forming property.

Conformational Sub-Space of Avirulent Toxins

Structural fluctuations (Cα-RMSF) were calculated between C2 and its avirulent mutants to know whether any constraints in conformational sub-dynamic space at a mutated residue (Table 4; Supplementary Fig. S5, S6). S350R (0.188–0.266) and E389K (−0.328 to −0.438) are structurally deviated from C2I with small extent in the side-chain of neighboring amino acids. We found no structural fluctuation in T349H and F398Y compared to C2I. E307S (0.266–0.477), E399C (1.374–1.233), F456H (0.359–0.712), and F456S (0.359–0.542) of C2II have shown conformational sub-dynamic flexibility in the side-chain of amino acids which are near to mutated site. However, no structural deviation is detected between F456A and C2II. This indicated that coevolutionary forces acting on the first mutated site may destabilize or stabilize the amino acid network of these mutants to withhold their native function.

Table 4 Impact of point mutation on the structural fluctuation (RMSF) and antigenic determinant sites in the mutated residues of C2I and C2II subunits
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Analysis of Immunogenic Nature of Avirulent Toxins

Antigenic determinants of C2 toxin were predicted and compared with its avirulent mutants (Table 4; Supplementary Fig. S7). It shows that E389K has more hydrophobicity at the residues near to mutated one. We found a more hydrophobic region in the S350R, indicating that it has more antigenic determinants in that region. The antigenic nature of F456S and C2II is almost similar to each other compared to other mutants. Antigenic determinants in E399C, F456A, and F456H predicted to be more than C2II, suggestive of their suitability as immunogens.

Discussion

A coordinated change of amino acid residues from more conserved to highly variable determines the molecular diversity of a protein during evolution (Engelhardt et al. 2011). Conserved domain-specific feature and a core of β-strands surrounded by α-helices reconcile a family-specific function of bacterial toxins and BADPRTs (Aktories et al. 2011; Chellapandi et al. 2013; Yeang and Haussler 2007). Phylogenetic analysis implied that C21 adaptor domain structure corresponds to similar domains of Ia and VIP2, and D1–D3 domain structure of C2II relates to PA and Ib in accordance with earlier studies (Aktories and Barth 2004; Tsuge et al. 2003, 2008). C2I and C2II share a common evolutionary origin like iota toxin, which might be evolved from BTA family at different evolutionary rates. Even if C2II is structurally and functionally similar to PA, D4 of C2II determines its receptor specificity and recognition (Barth et al. 2004; Varughese et al. 1999). Phylogenetic lineage-specific changes can resolve the functional specificity of C2 on its protein target, and to maintain a newly evolved functional feature from BTA family. NAD-binding core and catalytic mechanism of C2I resembles to C3, but its overall domain structure is dissimilar to C3. It may be a reason of that both of these toxins are belonged to two distinct families of ADP-ribosylation superfamily (Simon et al. 2014).

C2II/Toxin2A and C2I/Ia clusters are functionally evolved across the BTA family by duplication of these genes via Type I functional divergence and neutral selection. Gamma shape parameter (α) described the degree of heterogeneity rate across the BTA family. A protein fold may be reused during divergent or convergent evolution, which determines the functional range of a newly evolved protein (Gerlt and Babbitt 2001). Accordingly, NAD-binding core in C2 might be derived from C3 gene duplication by convergent evolution. Substrate-binding specificity slowly originated from sequence divergence within the BTA family by divergent evolution. The sequence-structure compatibility of a protein was analyzed by studying intergenic recombination events (Xia and Levitt 2002). C2 toxin gene locus is located in either plasmid or phage genome of C. botulinum. C2 toxin gene might be acquired by horizontal transfer mechanism that determines its virulence state. The number of sequence changes occurring along a lineage is more variable than expected with a constant rate of independent substitutions (Choi and Hannehall 2013). Evolutionary pattern analysis suggested that C2I and C2II have a common evolutionary pattern and evolved at the same rate. At the sequence level, it coordinates and stabilizes its prior structures (C2I–C2IIa) to have a function on the cell surfaces. The probable mutations being fixed in C2 structure depend on the residue position in accordance with Studer et al. (2013).

Coevolution sites are important constraints to understand the structural and functional evolution of a protein (Marks et al. 2012; Sandler et al. 2013). In our study, coevolution was an important force to be acted on the residues in α-helices, leading to change its local structural environment to β-sheets in coevolved pairs. Conformational flexibility in C2 structure was resulted by coevolved pairs as similar to C3 exotoxin (Prathiviraj et al. 2015). Even if these avirulent mutants have stable energetic structures similar to C2, a residue-coupling mutation may restrain the binding affinity to NAD+ or a protein substrate in accordance with earlier work on HIV-1 env protein (Travers et al. 2007). Since most of the functional sites are localized in its coiled and buried surface areas, correlated mutations in those sites may bring structural as well as functional changes (Aktories and Barth 2004; Barth et al. 1998; Blöcker et al. 2000; Sterthoff et al. 2010). Coevolution forces on the first residues may disorder or revolutionize its H-bonding pattern of coevolved pairs, leading to bring a radical change in H-bonding frequency. Therefore, coevolutionary constraints may prefer to stabilize its local structural environment and retained the non-toxic function.

The residues involving in catalytic and pathological mechanisms of the C2 are evolutionarily conserved across ADP-ribosyltransferases and also contributed in preserving the overall structural stability (Chavan et al. 1992; Han et al. 2001; Han and Tainer 2002; Kowarsch et al. 2010; Ménétrey et al. 2002, 2008). A structure–function link of ADP-ribosyltransferases was analyzed using several molecular constraints (Chellapandi 2014; Prathiviraj et al. 2015). Our study revealed that functional divergence of C2 structure was far slower than that of sequence. Thus, various structural constraints in the avirulent mutants of our study were detected to evaluate them as suitable avirulent toxins/immunogens.

Finding a suitable residue for replacing mutable one is an important measure to determine the structural and functional changes of a protein during the evolutionary process (Arenas et al. 2013). The structures of these avirulent mutants are stabilized by optimizing amino acid-atom potentials and torsion angle distribution (Gromiha et al. 2006). The SASA of these mutants are adjusted towards their buried surface area to hold the molecular function. Structure–function integrity may be retained by sustaining buried surface area and no H-bonding for detrimental binding. It suggested that avirulent mutants may analogously conserve their native-like structures. Secondary structural elements, particularly α-helices and β-sheets are not altered, but the loops/coils are reorganized from the native state, which was agreed with the previous work done by Sarabojia et al. (2005).

A conformational flexibility in fold states is a crucial constraint to design several proteins (Hammes et al. 2011; Ho and Agard 2010; Kuzmanic and Zagrovic 2010). Studies on protein folding rates are a measure to understand the pathogenic nature of mutants (Huang and Gromiha 2010, 2012). The fold rate on the structural classes may influence on the folding stability of these mutants compared to C2I. Our study suggested that there is no major impact of fold rates on the structural classes of protein folding process, but minor changes predicted in C2 mutants. It may be resulted due to the conserved NAD-binding β-sandwich toxin fold in wild-type C2 through all stages of the evolution (Blöcker et al. 2000; Han et al. 2001). Avirulent mutants with rapid folding rate do not require extensive evolutionary optimization, which was agreed with Riddle et al. (1997). Consequently, we revealed that avirulent mutants follow the protein folding rate from a common blueprint (β-sandwich toxin fold) of C2, which may suppose to carry out the fold recognition, process, stability and post translation modification in the expressed host. It also described that evolutionary constraint manifest the fast and efficient folding rates of these mutants and correlate with stability rather than contact order. Our hypothesis were correlated with earlier work (Monsellier and Chiti 2007).

Immunogens and vaccines can be designed by successful capturing near-native shifts in a protein backbone in response to mutational changes (Correia et al. 2014; Kuroda et al. 2012; Sevy and Meiler 2014). Avirulent mutants derived from C2I are structurally stable, and the resulted ensembles shown near native-like shifts in a protein backbone of C2I. F456S has shown a localized structural flexibility, indicating that its channel-forming property can be reduced when compared to C2II. The stability of a protein backbone structure and solvent accessibility can be affected by point mutations (Maguidet al. 2006; Sikosek and Chan 2014; Worth et al. 2009). Accordingly, we found several conformational flexibilities in the backbone structure of these mutants by mutational constraints in C2 toxin.

Conformational reorganization of binding sites is particularly important to design a protein-based vaccine (De Oliveira et al. 2011; Lapelosa et al. 2009; Topchiy et al. 2013). Herein, we demonstrated how single amino acid substitution can increase the conformational flexibility in the structures of mutants (Lapelosa et al. 2009; Lukman et al. 2010). Mutational constraints in conformational sub-dynamic space determine the substrate recognition across the BTA family and NAD-binding core among the BADPRTs superfamily. We also found coevolutionary constraints that are acting on the amino acid network of many avirulent mutants to stabilize their structures as well as withhold the native function in accordance with previous studies (Chellapandi et al. 2013; Chellapandi 2014; Prathiviraj et al. 2015; Travers et al. 2007). Geometric constraints that shape patterns of antigenic variation in Influenza H3N2 Hemagglutinin and bacterial toxins (Foley 2015; Meyer and Wilke 2015). Even a single amino acid substitution/mutation may bring antigenic evolution in many human viruses (Liang et al. 2010; Yoo and Deregt 2001). In our study, we found a drastic change in antigenic peptides predicted from avirulent toxins compared to C2 toxin. Thus, we suggested that evolution constraints-free immunogenic peptides can raise the humoral immunity in mammalian cells.

Conclusions

Our study is helpful in understanding the role of functional mutable residues in the ADP ribosyltransferase activity of C2I and channel and pore-forming properties of C2II on actin. Evolutionary genetic analyses described that the virulence state of C2 toxin is constrained by the residues present in conserved NAD-binding core, family-specific domain structure, and functional motifs. Structure–function integrity of C2 can be destabilized by single amino acid substitution in these regions. We have screened and selected nine avirulent mutants from C2 toxin showing more stability in their local structural environments with low evolutionary constraints. A coevolution force is also a concern of acting on stabilization of the local structural environment as well as conformational sub-dynamic space of C2 toxin upon a point mutation. Avirulent mutants have low NAD-binding specificity and pore/channel-forming capability by altering their H-bonding patterns and frequency. These mutants are structurally stable with low conformational flexibility, followed common blue print of rapid folding process. Antigenic determinants in avirulent toxins with lack of catalytic and pore-forming function is comparatively better than C2 toxin, suggestive of using them as immunogens for treating C. botulinum infected poultry and veterinary animals. Nevertheless, these avirulent toxins should be experimentally validated by protein engineering and cell culture techniques for successful protein-based subunit vaccines/immunogens candidates.