Mol Biol Rep (2012) 39:10243–10248 DOI 10.1007/s11033-012-1900-9
Structural insight into the mode of interactions of SoxL from Allochromatium vinosum in the global sulfur oxidation cycle Angshuman Bagchi
Received: 31 May 2012 / Accepted: 30 September 2012 / Published online: 7 October 2012 Ó Springer Science+Business Media Dordrecht 2012
Abstract Microbial redox reactions of inorganic sulfur compounds are one of the important reactions for the recycling of sulfur to maintain the environmental sulfur balance. These reactions are carried out by phylogenetically diverse microorganisms. The sulfur oxidizing gene cluster (sox) of a-proteobacteria, Allochromatium vinosum comprises two divergently transcribed units. The central players of this process are SoxY, SoxZ and SoxL. SoxY is sulfur compound binder which binds to sulfur anions with the help of SoxZ. SoxL is a rhodanese like protein, which then cleaves off the sulfur substrate from the SoxYZ complex to recycle the SoxY and SoxZ. In the present work, homology modeling has been employed to build the three dimensional structures of SoxY, SoxZ and SoxL. With the help of docking simulations the amino acid residues of these proteins involved in the interactions have been identified. The interactions between the SoxY, SoxZ and SoxL proteins are mediated mainly through hydrogen bonding. Strong positive fields created by the SoxZ and SoxL proteins are found to be responsible for the binding and removal of the sulfur anion. The probable biochemical mechanism of sulfur anion oxidation process has been identified. Keywords Docking simulations Environmental sulfur balance Homology modeling Sox operon Sulfur oxidation Electronic supplementary material The online version of this article (doi:10.1007/s11033-012-1900-9) contains supplementary material, which is available to authorized users. A. Bagchi (&) Department of Biochemistry and Biophysics, University of Kalyani, Nadia, Kalyani 741235, India e-mail:
[email protected]
Introduction Oxidation–reduction reactions involving environmental sulfur compounds maintain the balance of this important element in the atmosphere. The broad range of oxidation states of sulfur, such as, ?6 to -2, comes up with a number of important bioinorganic reactions which convert the sulfur from one form to the other. Such chemo- or photolithotrophic redox reactions of sulfur are mediated by phylogenetically diverse sets of microorganisms [1]. The typical sources of sulfur for these reactions are sulfide, thiosulfate, tetrathionate etc., producing reductants that are used for fixation of carbon-di-oxide or in the respiratory electron transfer chains [2]. The majority of the microorganisms capable of carrying out such redox reactions of sulfur are found to possess the gene cluster called the sox operon [3–8]. Nevertheless, the molecular mechanism of the sulfur oxidation process is poorly understood. The sox operon from a-proteobacteria Paracoccus pantotrophus (Para), Pseudaminobacter salicylatoxidans, Allochromatium vinosum (Avino) etc. contains two divergently transcribed transcriptional units comprising of genes soxSR and soxVWXYZABCDEFGH [3–8]. The proposed molecular mechanism of oxidation of thiosulfate anion in Para and Avino shows that thiosulfate gets coupled to the carboxy terminal cysteine residue of SoxY bound to SoxZ protein with the help of SoxXA. In the next step SoxB hydrolytically cleaves the SoxY-thiosulfate adduct to release a molecule of sulfate. The subsequent reactions differ in Para and Avino. In Para, Sox(CD)2 and SoxB help to recycle the SoxY protein by hydrolytically releasing a sulfate from SoxY. In Avino SoxCD is absent and a rhodanese like protein SoxL serves the purpose of recycling of SoxY protein. The organism Avino has a very vital in role maintaining the ecological balance by removing sulfide and thereby recycling elemental
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sulfur from environments which are deemed ‘sulfide-polluted’. Thus the organism is said to possess bio-catalytic properties which contribute to the continuation of the global sulfur cycle [9]. Para does not have SoxL [10]. SoxY, SoxZ and SoxL of Avino are proteins with 154, 104 and 224 amino acid residues, respectively. SoxY is a sulfur covalently binding protein while SoxZ is a sulfur compound chelating protein. During the sulfur oxidation process, SoxY and SoxZ combine with each other to form a complex (SoxYZ complex). Sulfur anion (thiosulfate for example) combines with the carboxy terminal cysteine residue of SoxY and forms an adduct [3–8, 10]. The SoxL protein in Avino then comes into play and removes the sulfur substrate from SoxY of SoxYZ complex [8, 10]. However, to date the detailed structural information regarding the interactions between these proteins has not been fully understood. In the present context, the aim of the study is to understand the structural bases of the involvements of these proteins in sulfur oxidation cycle. In the present study, the three dimensional structures of SoxY, SoxZ and SoxL from Avino obtained by homology modeling have been described. Molecular docking simulations have been performed in order to find out the possible modes of binding of these proteins. Binding sites of SoxY, SoxZ and SoxL have been predicted and analyzed. These studies provide a detailed structural insight into the plausible molecular mechanism of the involvements of these proteins in the global sulfur oxidation reaction cycle. As this is the first report regarding the structural bases of the involvements of SoxY, SoxZ and SoxL from Avino in the process of biochemical oxidation of sulfur anions, these results may contribute towards the understanding of the molecular mechanism of sulfur anion oxidation by these ecologically important microbial species.
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like protein) from Mycobacterium tuberculosis (PDB code: 3HWI) with sequence identity of 34 %. When the amino acid sequences of the proteins were used again to perform sequence-structure alignment using FUGUE [13], the same pdbs as obtained by BLAST search were obtained. The Y and Z chains of 2OXG were separated and used to build the models of SoxY and SoxZ proteins of Avino. The model of SoxL was built using 3HWI as template. The homology modeling was performed using the software suite present in the Homology module of Insight II (Accelrys, San Diego, CA, USA). The modeled structures were then superimposed separately on each of the crystal templates without altering the coordinate systems of atomic positions in the respective templates (Y chain of 2OXG for SoxY, Z chain of 2OXG for SoxZ and 3HWI for SoxL). The root mean square ˚ deviations (RMSD) for the superimpositions were 0.5 A ˚ for both SoxY and SoxZ and 0.9 A for SoxL. The mode of the superimposition of SoxY onto its crystal template was presented in Fig. 1. Since the mode of superimpositions were similar for SoxZ and SoxL therefore only one (that of SoxY onto its crystal template) was presented. The models of the proteins were then energy minimized in two steps. In the first step the modelled structures were minimized without restraints. In the second step the energy minimizations were done by fixing the backbones of the modeled proteins to ensure proper interactions. All energy minimizations were done using conjugate gradient (CG) with CHARMM force fields [14] using the program DISCOVER until the structures reached the final derivative of 0.001 kcal/mol. The results were further validated using the consistent valance force field (CVFF) [15]. Same results were obtained as observed in case of CHARMM force fields.
Materials and methods Sequence analysis and homology modeling of monomeric SoxY, SoxZ and SoxL proteins The amino acid sequences of SoxY, SoxZ and SoxL proteins of Avino were obtained from Entrez database. The Accession nos. of the proteins are DQ441405 for SoxX and SoxL and DQ441406 for SoxZ. These amino acid sequences were used separately to search the Brookhaven Protein Data Bank (PDB) [11] using the software BLAST [12] to find suitable templates for homology modeling. The search results for SoxY and SoxZ picked up the X-ray crystal structure of the SoxYZ complex from Paracoccus denitrificans (PDB code: 2OXG; Y chain for SoxY and Z chain for SoxZ) with 50 % sequence identity. For SoxL the best template was found to be the crystal structure of probable thiosulfate sulfurtransferase Cysa2 (rhodanese-
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Fig. 1 (For web version): Superimposition of the a-carbon backbones of SoxY (black) on the Y chain of 2OXG (red). (Color figure online)
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Validation of the models
Results
Regarding the main chain properties of the modeled proteins, no considerable bad contacts nor Ca tetrahedron distortions nor hydrogen bond (H-bond) energy problems were found. There were no side chain distortions as observed by measuring the side chain torsion angles. The Z scores calculated using the software PROSA 2003 [16] showed that the predicted homology models were well inside the range of typical native structures [17]. The residue profiles of the three dimensional models were further checked by VERIFY3D [18]. PROCHECK [19] analyses were performed in order to assess the stereochemical qualities of the models and Ramachandran plots [20] were drawn. No residues were found to be present in the disallowed regions of the Ramachandran plots.
Description of the structure of SoxY
Molecular docking simulations In order to study the interactions between SoxY and SoxZ proteins as well as between SoxL and the SoxYZ protein complex, first the models of the SoxY and SoxZ proteins were docked using the software GRAMM [21]. The docking of the proteins was also performed with DOT [22] and ZDOCK [23], using the ClusPro server [24] in order to get a comprehensive result. The docked structure of the SoxYZ complex that yielded the best score among all the other possible docked structures was selected and analyzed visually using Insight II. The complex of SoxYZ was then energy minimized as per the protocol mentioned previously in the ‘‘Sequence analysis and homology modeling of monomeric SoxY, SoxZ and SoxL proteins’’ section. The resulting energy minimized structure of the SoxYZ complex was used to dock with SoxL protein using the aforementioned software tools. The resulting best structure, that had the best score among all the possible docked structures, was selected and analyzed visually using Insight II. Again the SoxYZL protein complex was subjected to energy minimization as per the previously mentioned protocol in the ‘‘Sequence analysis and homology modeling of monomeric SoxY, SoxZ and SoxL proteins’’ section.
Calculation of protein–protein interactions To find out the interactions between the SoxY, SoxZ and SoxL proteins, What If software package [25] as well as the Biopolymer module of Insight II were used. These programs calculate the interactions between two groups by measuring the distance between them.
The modeled structure of SoxY is a 154 amino acid residue long protein. The predicted structure is similar to the SoxY protein from the SoxYZ complex from Paracoccus denitrificans (PDB code: 2OXG; Y chain for SoxY). It starts with a helix (amino acid residues 4–12), followed by a short turn region (amino acid residues 15–18). The remaining part is made up of b-strands interspersed with turn regions. At the middle of this protein there is a fourstranded anti-parallel b-sheet (amino acid residues 74–83, 87–94, 102–107 and 114–122). The structure is presented in Fig. S1. Description of the structure of SoxZ The model of SoxZ protein consists of 104 amino acid residues. The predicted structure is similar to the SoxZ protein from the SoxYZ complex from Paracoccus denitrificans (PDB code: 2OXG; Z chain for SoxZ). The protein is made up of b-strands (amino acid residues 6–10, 13–21, 44–52, 53–60, 70–77 and 84–90), which produce a six-stranded anti-parallel b-sheet connected by loops. Figure S2 shows the structure of the modeled protein. Description of the structure of SoxL The modeled structure of SoxL is a 224 amino acid residue long protein. The predicted structure is similar to the crystal structure of probable thiosulfate sulfurtransferase Cysa2 (rhodanese-like protein) from Mycobacterium tuberculosis (PDB code: 3HWI). The protein has a rhodanese like signature sequence. It starts with a parallel b-sheet linked by a helix. The remaining part is made up of b-strands interspersed with helices and turn regions. The structure is presented in Fig. S3. Interaction of SoxY with SoxZ In order to find the interactions between the proteins the three dimensional coordinates of the proteins were docked by the software tools GRAMM, DOT and ZDOCK. All the software tools produced somewhat similar results. This shows the consistency of the docking experiment. SoxY and SoxZ are found to interact strongly with each other. The protein–protein interface is found to mainly contain the polar amino acid residues. The interior of the complex is made up of hydrophobic amino acids. There are extensive H-bonding interactions involving both the main and the side chains of the two protein molecules. The SoxYZ complex is also stabilized by ionic interactions. SoxY
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software tools produced somewhat similar results. This shows the consistency of the docking experiment. The SoxL protein binds to SoxYZ complex. The protein binds to the complex by H-bonding as well as by ionic interactions. Interestingly, the sulfur anion binding domain of SoxY is found to be surrounded by the side chains of six positively charged amino acid residues of SoxL (Arg153, Arg160, Lys173, Lys203, Lys205, Arg208) replacing the four residues of SoxZ that were present in the SoxYZ complex to facilitate sulfur anion binding. The stronger positive environment created by these amino acid residues would be required to break the covalent bond between the Sc atom of the Cys152 of SoxY in the SoxYZ complex. Besides that, the attractive force of SoxL on sulfur anion is more than that furnished by SoxZ in the SoxYZ complex due to the creation of a stronger positive field by SoxL as more positively charged residues (six positively charged residues of SoxL in place of four positively charged residues of SoxZ) surround the sulfur anion binding domain of SoxY in the SoxY–SoxZ–SoxL protein complex. Figure 3 depicts the interactions of the proteins.
Fig. 2 (For web version): Interactions of SoxY and SoxZ. The active site cysteine residue (Cysteine152) is highlighted. The other residues of SoxZ present near the active site cysteine are shown
protein of Avino contains a sulfur anion binding signature GGCGG sequence at the carboxy terminal region of the protein where the free thiol group of the cysteine 152 residue (presented in bold above) binds the sulfur anion (thiosulfate). Interestingly, amino acid residues from the sulfur anion binding motif of SoxY do not take part in this interaction scheme. The sulfur anion binding region of SoxY forms a cavity which is surrounded by four positively charged amino acid residues (Lys31, Lys58, Lys67 and Lys74) of SoxZ. This positive environment in the complex may help to drag the sulfur anion near the active site of SoxY which then covalently binds the anion. Figure 2 represents the binding of the two proteins. The active site cysteine residue (Cys152) is found to be freely available in the SoxYZ complex. Interaction of SoxL with SoxYZ complex In order to find the interactions between the proteins the three dimensional coordinates of the proteins were docked by the software tools GRAMM, DOT and ZDOCK. All the
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Fig. 3 (For web version): Interactions of SoxYZ complex with SoxL. The active site cysteine residue (Cysteine152) is highlighted. The other residues of SoxL present near the active site cysteine are shown
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Discussion In this study, an attempt has been made to elucidate the structural basis of the involvements of SoxY, SoxZ, and SoxL in the binding and release of sulfur anion during the global sulfur oxidation reaction cycle. For that matter the three-dimensional structures of the proteins SoxY, SoxZ, and SoxL have been built and analyzed. The geometry of the sulfur anion binding region of SoxY has also been established. The putative mode of binding of SoxY with SoxZ has been analyzed and the possible molecular basis of binding of sulfur anions in general has been described. In the crystal template (2OXG) for SoxYZ protein complex, it was observed that the side chain of a cysteine residue from SoxZ was inserted in the core of the protein complex [26]. In case of the interactions between SoxY and SoxZ proteins from Avino Cys59 was found to be inserted in the core of the protein complex. Overall, the SoxYZ protein complex in 2OXG is stabilized by hydrogen bonding interactions as observed in case of SoxYZ complex from Avino. The plausible molecular biology for the formation of the hetero-trimeric complex of SoxY–SoxZ– SoxL has also been demonstrated to predict the biochemical pathway of sulfur anion binding and release via these proteins in the global sulfur anion oxidation reaction cycle as the SoxYZ complex is the central player in the oxidation of sulfur anions. Since there have been no previous reports regarding the structural biology of these proteins, results from this study may shed light to understand the three dimensional structures of SoxY, SoxZ, and SoxL as well as to elucidate the structural basis of the molecular functions of these proteins. This model provides a rational framework for designing experiments to determine the contribution of the various amino acid residues in these proteins to predict the molecular basis of their interactions both among themselves as well as with various sulfur anions.
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15. Acknowledgments The help and support rendered by Prof. Tapash Chandra Ghosh of Bioinformatics Center, Bose Institute, AJC Bose Centenary Building, P1/12 CIT Scheme VII M, Kolkata 700 054, India are duly acknowledgement here. The author is also thankful to the DBT sponsored Bioinformatics Infrastructure Facility in the Department of Biochemistry and Biophysics, University of Kalyani for the necessary support. Finally, the author would like to thank the anonymous referee for the valuable comments to make the manuscript better.
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