Adaptive evolution in the toxicity of a spider's venom enzymes

Abstract

Background: Sphingomyelinase D is the main toxin present in the venom of Loxosceles spiders. Several isoforms present in these venoms can be structurally classified in two groups. Class I Sphingomyelinase D contains a single disulphide bridge and variable loop. Class II Sphingomyelinase D presents an additional intrachain disulphide bridge that links a flexible loop with a catalytic loop. These classes exhibit differences in their toxic potential. In this paper we address the distribution of the structural classes of SMase D within and among species of spiders and also their evolutionary origin by means of phylogenetic analyses. We also conducted tests to assess the action of natural selection in their evolution combined to structural modelling of the affected sites.

Results: The majority of the Class I enzymes belong to the same clade, which indicates a recent evolution from a single common ancestor. Positively selected sites are located on the catalytic interface, which contributes to a distinct surface charge distribution between the classes. Sites that may prevent the formation of an additional bridge were found in Class I enzymes.

Conclusions: The evolution of Sphingomyelinase D has been driven by natural selection toward an increase in noxiousness, and this might help explain the toxic variation between classes.

Fig. 1

Partial multiple alignment of amino acid sequences of SMase D. In this analysis, sites Cys⁹⁶ and Cys¹⁰² correspond to Cys⁵¹ and Cys⁵⁷, respectively, and form a disulphide bridge in Class I and Class II isoforms. The sites Cys⁹⁸ and Cys²⁶⁴ (corresponding to Cys⁵³ and Cys²⁰¹) form an additional bridge in SMase D Class II members

Fig. 2

Phylogenetic analysis of SMase D sequences by ML method. Phylogenetic tree based on the ML method (lnL = −46,243.0294). A GTR model was chosen (BIC score = 72,153; lnL = −33,053.48462). Gamma distribution (G parameter = 2.5232). The rate variation model allowed certain sites to be evolutionarily invariable (I). Loxosceles SMase D Class I isoforms are indicated in clades L and G (red)

Fig. 3

Phylogenetic analysis of SMase D sequences by nucleotide model. Bayesian inference tree based on the nucleotide model. A GTR model was chosen (BIC score = 72,153; lnL = −33,053.48462). Gamma distribution (G parameter = 2.5232). The rate variation model allowed certain sites to be evolutionarily invariable (I). Posterior probabilities are indicated. Loxosceles SMase D Class I isoforms are indicated in clades L and G (red)

Fig. 4

Phylogenetic analysis of SMase D sequences by codon model. Bayesian inference tree based on the codon model. A GTR model was chosen (BIC score = 72,153; lnL = −33,053.48462). Gamma distribution (G parameter = 2.5232). The rate variation model allowed certain sites to be evolutionarily invariable (I). Posterior probabilities are indicated. Loxosceles SMase D Class I isoforms are indicated in clades L and G (red)

Fig. 5

Estimates of ω ratios for sites in the SMase D enzymes. Positively selected sites (ω > 1) for both SMase D classes are indicated

Fig. 6

Statistical analysis of SMase D classes for each site under positive selection. The absolute frequency of amino acids found in each positively selected site is indicated for Classes I and II. Nonpolar, basic polar, neutral polar and acidic polar amino acids are indicated. The p-values obtained by the Wilcoxon test are shown

Fig. 7

a Statistical analysis of SMase D classes for sum of sites under positive selection. The absolute frequency of summed amino acids is indicated for Classes I and II. Nonpolar, basic polar, neutral polar and acidic polar amino acids are indicated. The p-values obtained by the Wilcoxon test and t test are shown. b Distribution of nonpolar + basic and neutral and acidic residues in Class I and Class II members

Fig. 8

Structural interpretation of the positively selected sites. a Cartoon representation of the structural comparison between Class I and II SMases D. The fully conserved catalytic histidines (H12 and H47) and the three acidic residues (E32, D34 and D91) involved in the metal ion coordination are shown as sticks, with carbon atoms in green. The seven positively selected sites are shown as sticks and balls, with carbon atoms in yellow. The residues depicted in the positive sites correspond to those of SMase I from L. laeta, and the sequence numbering is also based on this molecule according to PDB entry 1XX1 (Murakami et al., 2005). The cartoon representation is coloured according to the secondary structure elements and the flexible loop F related to the second S-S bond found uniquely in Class II members (in orange, Class I; in red, Class II). The variable loop E is cyan and blue for Classes I and II, respectively. b Schematic representation of a Class I SMase D highlighting all of the positively selected sites for this class using the same colour pattern

Fig. 9

Electrostatic surface charge distribution of Class I and II SMases D highlighting the catalytic interface. The ellipses indicate the active-site pocket. a Class I; b Class II