Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

Abstract

Background: The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms.

Results: The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies.

Conclusion: The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials.

Figure 1

Biosynthetic path of AI-2 utilizing LuxS to detoxify SRH.

Figure 2

Schematic representation of the triangular (isosceles) orientation of the active site (I/II) and apex clusters. The active site clusters I and II (shown by light green oval shaped region) and the mini-triad (shown as a mauve triangle) at the apex of the dimer interface for the protein LuxS (Average edge lengths are given); the protein backbone shown in tube representation with the two chains coloured differently.

Figure 3

All Interface amino acid clusters in one representative member from each class (I–VI). (a) CLASS I (H. influenzae), (b) CLASS II (H. pylori), (c) CLASS III (B. subtilis), (d) CLASS IV (D. radiodurans), (e) CLASS V (V. cholerae), and (f) CLASS VI (L. johnsonii), at I_min = 6%. The two subunits of the LuxS dimer are represented in different colours (chain A in white and chain B in green) by new cartoon representation. The cluster forming residues are shown in van der Waal's representation (Blue spheres and red spheres represent the residues from chain A and chain B respectively).

Figure 4

Triangular pattern at the dimer interface. The pattern comprises of the two active sites clusters I and II and the apex cluster at the top vertex of LuxS. (a) Isosceles triangular pattern of the amino acid residues in the interface clusters (cluster I, II and apex cluster) well-organized at the dimer interface for m1eco. The edges of the triangle are calculated to be 31.66, 31.42 and 13.09 Å for active-site cluster I, apex cluster, and active site cluster II moving clockwise, (b) Isosceles triangular pattern of the amino acid residues in the interface cluster is distorted at the dimer interface for the probiotics and some extremophiles, as shown here for the probiotic m1lac. The edge between apex cluster and active site cluster II is 31.33 Å but active-site cluster I is missing from the interface cluster as the three His (chain A) have moved away from Phe (chain B) by 15 Å as compared to 8 Å distance between Phe (chain B) and the three His (chain A). The backbone is represented by transparent new cartoon and the amino acid residues at the vertices of the triangle as van der Waal's spheres; each monomer and its amino acid residues coloured differently. Residues from chain A are coloured Blue and those from B are coloured Red. An imaginary line is drawn across the interface in yellow and the distances between His 58 (CB) and Phe 7 (CB) are approximately given for m1lac.

Figure 5

Interface signature motif. The signature motif for (a) CLASS V (m1vib, m1clo, m1pyo, and m1shi), and (b) CLASS VI (m1lac, m2lac, m3lac, and m1bfi); based on superposition of all the LuxS structures belonging to a particular class and prediction of structurally superposing residues (by manual inspection) as depicted pictorially to form a motif at I_min = 6%. The superposed backbone structures are represented as transparent new cartoon and the interface cluster residues forming the motif are shown as van der Waal's spheres, specific colour represents residues from a specific protein within the class. The different regions are marked and labelled with the corresponding residues in different colours; red/pink indicates completely/partially conserved residues respectively within the class, whereas violet indicates no conservation within the class. The completely/partially conserved residues are in bold/bold italics respectively. The residues coming from only one subunit are underlined in blue (chain A) and mauve (chain B).

Figure 6

Apex cluster at the dimer interface. The triads formed from sequentially apart residues (x (A/B), x+2 (A/B), x+4 (B/A), where x is an arbitrary sequence number) at the top vertex found for all LuxS proteins under study (except for Bacillus sp. LuxS). The triad for 1j6x (class II) is depicted here; the backbone is represented as new cartoon and each monomer and the residues coming from it are coloured differently.

Figure 7

Representation of Multiple sequence alignment (using ClustalW) within class IV. The "persistent hub" and the four connecting residues are highlighted in the figure with coloured lines; the green arrow indicates the "persistent hub" and the red arrows indicate the connecting residues and are numbered arbitrarily from 1 to 4. Residue 2 and 3 are completely conserved within the class whereas residue 4 is partially conserved. However the "persistent hub" and the residue 1 are completely conserved within class IV excluding m1psy. The region marked in the orange box indicates the residues present in the mini-triad for class IV; the mini-triad sequence being absent from m1the.

Figure 8

Frequency of occurrence (as shown in the Y-axis) of the predicted sequentially non-conserved mini-triad motifs (details shown in the X-axis) in a dataset of 202 proteins from the LuxS family. 'X' indicates any amino acid residue.

Figure 9

Representation of the "persistent hub" present at I_min = 12% for 1inn(class IV) LuxS. The active site cluster, the hub and their connecting residues are represented by van der Waal's spheres. The hub is coloured purple and the connecting residues are coloured yellow. Blue spheres depict residues from chain A and red represents those from chain B in the Active site cluster. The protein backbone is shown in new cartoon representation with the two subunit coloured differently. An imaginary line is drawn across the interface in orange.

Figure 10

(a-f): Connectivity representation (drawn using Graphviz[19]) of the residues in the largest interface cluster as obtained from graph theoretical analysis. One representative member is chosen from each class: (a) class I (1j6w), (b) class II (1j6x), (c) class III (1j98), (d) class IV (1inn), (e) class V (m1shi), and (f) class VI (m1lac) respectively. The residues from chain A are coloured orange and those from chain B are coloured yellow.