Abstract: In this study, we have tried to elucidate the structural basis of the involvements of SoxA and SoxX in the binding and in electron transport during oxidation of sulfur anions via the novel global sulfur oxidation cycle. SoxA is known to be a di-heme cytochrome c protein, which helps in binding sulfur anions with SoxY. SoxX, a mono-heme cytochrome c protein, binds to SoxA forming a complex, which helps in efficient transport of electrons during oxidation of sulfur anions. We employed homology modeling to construct the three-dimensional structures of the SoxA and SoxX from Pseudaminobacter salicylatoxidans and established the geometry of the cytochrome c region and that of the active site of SoxAX complex. The docking studies with SoxA and SoxX allowed us to identify the details of SoxA-SoxX interactions. The structural basis of the formation of the hetero-dimeric complex of SoxA-SoxX were also demonstrated to predict the biochemical pathway of sulfur anion binding and transport of electrons via these proteins in the novel global sulfur cycle as SoxAX complex is the initiator of the sulfur anion oxidation pathway. Since there have been no previous reports regarding the structural biology of these proteins, results from this study will be important for the understanding of the three dimensional structures of SoxA and SoxX as well as to elucidate the structural basis of their mode of actions.
Introduction
Microbial redox reactions of sulfur are mainly responsible for cycling of this element in the environment to maintain environmental sulfur balance. Sulfur has a unique range of oxidation states that varies from +6 to -2 and as a result several important biological processes involving transformations of sulfur from one form to other have been evolved. Sulfur based chemo- or photolithotrophy is one of such processes in which electron transfer from reduced sulfur compounds is used by phylogenetically diverse prokaryotes (Friedrich, 1998; Fowler and Crundwell, 1999; Okabe et al., 1999). Various sulfur anions such as, sulfide, polysulfide, thiosulfate, polythionates, sulfites as well as elemental sulfur are the different forms of sulfur in the environment (Le Faou et al., 1990), which serve as the electron donors in the process of respiration or photosynthesis of the sulfur oxidizing prokaryotes. Only little is understood about the molecular mechanism of this oldest known process.
Recent studies with both chemo- and photolithotrophic α-Proteobacteria, such as Paracoccus pantotrophus (Para), Pseudaminobacter salicylatoxidans (KCT), revealed that multiple-gene cluster, shxVW (soxVW) and soxXYZABCDEFGH, is associated with metabolism sulfur anions (Deb et al., 2004; Mukhopadhyaya et al., 2000). SoxXA, SoxYZ, SoxB and SoxCD are required for sulfur-dependent cytochrome c reduction. The eight-electron oxidation of a molecule of thiosulfate is governed by cytochrome c complex multienzyme system (TOMES) encoded by soxXYZABCD (Rother et al., 2001; Friedrich et al., 2001).
It is to be noted that Pseudaminobacter salicylatoxidans is also involved in the oxidative hydrolysis of highly toxic halo-alkanes SoxA and SoxX of KCT are proteins with 286 and 154 amino acid residues respectively. SoxA and SoxX are a di-hem (Mukhopadhyaya et al., 2000) and mono-heme cytochrome c proteins respectively. Sequence analysis of the SoxX protein from some other organisms like Paracoccus pantotrophus reveal that it contains a mono-heme subunit (Rother et al., 2001; Friedrich et al., 2001). During the sulfur oxidation process SoxA and SoxX combine with each other to form a complex (SoxAX complex). The complex accepts electrons from sulfur anions and thereby facilitates the binding of sulfur anions to a conserved cysteine residue of SoxY to form SoxY-thiocysteine-S-sulfate, the first covalently bonded sulfur adduct during sulfur anion oxidation by sox operon (Friedrich et al., 2001; Mukhopadhyaya et al., 2000). However to date there are very few reports regarding the structural aspect of the involvements of these proteins in the global sulfur cycle. In the present study our aim is to understand the structural basis of the interaction of SoxA and SoxX from KCT to investigate the molecular mechanism of sulfur anion oxidation via sox operon. We describe the three-dimensional structures of these proteins obtained by homology modeling. We have docked the two proteins in order to investigate their favorable binding modes. Molecular dynamics simulations have been performed on the protein complex in order to properly find out the amino acid residues involved in the protein-protein interaction. Binding interactions between SoxA and SoxX have been analyzed to predict the possible molecular mechanism of electron transport during sulfur anion oxidation. These studies provide detailed structural information regarding the molecular biochemistry of the binding of SoxA with SoxX from KCT. As this is the first report regarding the structural aspects of the interaction of SoxA and SoxX from KCT in the process of oxidation of sulfur anions via sox operon, our studies are expected to contribute towards the understanding of the molecular details of the biochemical pathway of sulfur anion oxidation by these ecologically important microorganisms.
Materials and Methods
Sequence Analysis and Homology Modeling of Monomeric SoxA and SoxX
The amino acid sequences of SoxA and SoxX of KCT were obtained from Entrez
database (Accession No. CAB94219 and CAH59732 respectively). These amino acid
sequences were used separately to search Brookhaven Protein Data Bank (PDB)
(Berman et al., 2000) using the software BLAST (Altschul et al.,
1990) for finding suitable template for homology modeling. The BLAST search
result of SoxA and SoxX showed 54 and 50% sequence similarities respectively
with the X-ray crystal structure of SoxAX complex from Rhodovulum sulfidophilum
(pdb code: 1H33). In the complex 1H33 A and B chains referred
to the crystal structures of SoxA and SoxX respectively. The models of SoxA
and SoxX were built using the A and B chains respectively of the crystal 1H33
as templates. Homology modeling was performed using the program Homology of
Insight II (Accelrys, San Diego, USA) on a Silicon Graphics Indigo II workstation.
Modeled structures were then superimposed separately on each of the crystal templates without altering the coordinate system of atomic positions in the respective templates (A and B chains of 1H33 for SoxA and SoxX, respectively). The mean r.m.s deviations for the superimpositions were 0.4Å and 0.3Å for SoxA and SoxX respectively on their corresponding crystal templates (Fig. 1 and 2, respectively). Short contacts and bad regions were rectified manually by Insight II.
| Fig. 1: | Superimposition of the α-carbon backbones of SoxA (Black) on the A chain of 1H33 (Red) |
| Fig. 2: | Superimposition of the α-carbon backbones of SoxX (Black) on the B chain of 1H33 (Red) |
The models were then energy minimized fixing the backbones to ensure proper interactions. Conjugate Gradient (CG) method was employed for minimization with the Consistent Valence Force Field (CVFF) (Dauber-Osguthorpe et al., 1988) using the program DISCOVER3 of Insight II until all the structures reached the final derivative of 0.001 Kcal/mol and were validated using VERIFY3D (Eisenberg et al., 1997). PROCHECK (Laskowski et al., 1993) analysis was performed in order to assess the stereo-chemical qualities of the three dimensional models and Ramachandran plots (Ramachandran and Sashisekharan, 1968) were drawn. No residues were found to be present in the disallowed regions of the Ramachandran plot.
The accessible surface areas of the predicted cytochrome c motifs of the final structures of SoxA and SoxX were calculated with the program NACCESS (Hubberd, 1992).
Docking of Iron-prophyrine Complex with SoxA and SoxX
To study the interactions involved in the binding of the Iron-Prophyrine
complex with SoxA and SoxX, the cytochrome c motifs of the proteins were superimposed
on the corresponding cytochrome c motifs of SoxA and SoxX of 1H33 in
presence of the Iron-Prophyrine complex. The r.m.s deviations of superimpositions
were 0.3 Å for the two cytochrome c motifs of SoxA and 0.4 Å for
the cytochrome c motif of SoxX on the corresponding regions in the crystal templates.
The complex was subsequently merged with the model to mimic a model- Iron-Prophyrine
complex.
Docking of SoxA and SoxX
To study the interactions involved in the binding of SoxA and SoxX, the
modeled proteins were superimposed separately on the A and B chains of the crystal
structure of 1H33. The models were subsequently merged with the crystal
templates and the crystals were removed to produce a modeled SoxAX complex.
The docked structure of the SoxAX complex, thus obtained, was subjected to energy
minimization using the program DISCOVER3 of Insight II initially by Steepest
Descent (SD) and then by CG with CVFF keeping the backbone atoms fixed, until
model reached the final derivative of 0.001 kcal/mol.
Molecular Dynamics Calculations
Molecular Dynamics (MD) simulations were performed on the docked structure
to predict the favourable binding interactions. The docked structure was solvated
with an average of 2000 simple point charges (Berendsen et al., 1981)
water molecules. The system was minimized initially keeping the water and the
backbones of the proteins fixed. In the next step of minimization, the protein
complex was kept fixed and the water molecules were allowed to move. The first
few rounds of minimizations were performed by steepest descent (SD) method and
then CG method was employed. The minimized system was equilibrated for a period
of 10 ps with positional restraints. Then a 120 ps MD run was performed without
restraints. Weak coupling of the protein to a solvent bath of constant temperature
(300 K) and constant pressure (1 bar) was maintained with a coupling of 1.0
ps. For all energy minimizations and MD simulations GROMACS molecular simulation
package (Lindahl et al., 2001) was used. All the structures were finally
analyzed by PROCHECK.
Results and Discussion
Description of the Structure of SoxA
The modeled structure is a 286 amino acid residue long protein. The predicted
structure is similar to X-ray crystal structure of SoxA protein from Rhodovulum
sulfidophilum (Bamford et al., 2002). The first 27 residues show
the characteristic feature of a signal peptide. This was predicted by PrediSi
(Hiller et al., 2004). The protein contains two domains. The first domain
(amino acid residues 1 to 143) starts with a helix (amino acid residues 9 to
15) followed by a three-stranded parallel β-sheet (amino acid residues
33 to 35, 38-40, 43 to 44). The first cytochrome c motif consists of a helix
(amino acid residues 100 to 104) followed by a short bend, which ends in another
helix (amino acid residues 107 to 110). The central core of the protein is made
up of helices interspersed with β-sheets connected among themselves by
short turns and bends. The second cytochrome c motif (amino acid residues
202 to 206) is also made up of a helix, which is connected to a β-sheet
by short turns and bends. The rest part of the protein is made up of helices
and sheets connected among themselves by loops and turns. The structure is presented
in Fig. 3.
Description of the Structure of SoxX
The model consists of 154 amino acid residues. The predicted structure is
similar to the SoxX protein from Rhodovulum sulfidophilum. The first
21 amino acids show the presence of a typical signal peptide. The protein is
made up of helices and sheets connected by loops and bends. The cytochrome c
motif consists of a bent helix (amino acid residues 61 to 67) followed by loops
and bends and ends in another helix (amino acid residues 87 to 90). The rest
part of the protein is made up of helices (amino acid residues 93 to 102 and
139 to 147) and sheets (amino acid residues 126 to 127 and 132 to 133) connected
by loops and bends. Figure 4 represents the structure of the
modeled protein.
Comparison with Other Cytochrome C Proteins
SoxX has strong structural similarities with other known cytochrome
c proteins viz., Bacillus pasterii (1C75) (Benini et al.,
2000), Thermus thermophilus (1C52) (Than et al., 1997),
Desulfovibrio vulgaris (1C53) (Nakagawa et al., 1990),
Monoraphidium braunii (1CTJ) (Frazao et al., 1995). Superimpositions
of the cytochrome c motif of SoxX with the corresponding motifs in 1C75,
1C52, 1C53 and 1CTJ give r.m.s deviations of 0.8, 0.9,
0.8 and 0.7 Å, respectively showing that the overall structure of SoxX
is similar to these well-known cytochrome c family proteins (Fig.
5).
Residue-wise accessibility of the cytochrome c motifs of both the proteins were determined and shown in Table 1. The Table 1 shows that out of a total of 10 residues 3 residues are above 30% solvent exposed. This predicts that the cytochrome c motifs are buried in the protein core. Similar results were also shown for other proteins having cytochrome c motif (Hasegawa et al., 2000).
| Table 1: | Residue-wise absolute and % accessibilities of the cytochrome c motifs of SoxA and SoxX of KCT |
| *Accessibilities are in square angstrom, *Percent accessibilities are shown in parenthesis | |
| Fig. 3: | Ribbon representation of modeled SoxA. -helices (Red and Yellow) and -sheets (Cyan) are shown as helices and ribbons, respectively. The rest are shown as loops. The figure was prepared by MOLSCRIPT (Kraulis, 1991) |
| Fig. 4: | Ribbon representation of modeled SoxX. -helices (Red and Yellow) and -sheets (Cyan) are shown as helices and ribbons respectively. The rest are shown as loops. The figure was prepared by MOLSCRIPT (Kraulis, 1991) |
| Fig. 5: | Superimposition of the -carbon backbones of the cytochrome c motif of SoxX (red) on the similar motifs of 1C75 (violet), 1C52 (cyan), 1C53 (black) and CTJ (mauve). The mode of superimposition is similar for the cytochrome c motifs of SoxA; therefore only one is shown for clarity |
| Fig. 6: | Electrostatic potential map of the surface of the SoxAX complex showing the positively charged substrate active funnel |
SoxA- Iron Porphyrine Interaction
In the crystal structure of 1H33 (A chain) the first heme group is
covalently bound to the protein matrix through thioether bonds, between CysA76
and CysA114 Sγ atoms and the heme Cα positions 2 and 4, respectively
(Bamford et al., 2002). Superimposition of the first cytochrome c motif
of SoxA on the same motif of 1H33 produced an r.m.s deviation of 0.3
Å. The distance of the Sγ atoms of
| Table 2: | Residues of SoxA and SoxX involved in ionic interaction in the SoxAX complex |
Cys100 and Cys138 of SoxA (similar to the CysA76 and CysA114, respectively of 1H33) and the heme Cα atoms at positions 2 and 4, respectively are found to be 1.47 and 1.76 Å. This implies these groups are close enough to form covalent bonds with the protein matrix as observed in case of 1H33.
The crystal structure of 1H33 (A chain) also shows that the Fe atom of the heme group of the iron-porphyrine complex is coordinated to the Nε atom of HisA80 (Bamford et al., 2002). In SoxA the distance between the Nε atom of His104 and the Fe atom of the iron-porphyrine complex is 2.34, which is close enough to form a coordinate covalent bond with the Fe atom.
Similarly, for the second cytochrome c motif of SoxA, Cys202 and His206 are involved in the interactions with the iron-porphyrine complex analogus to the CysA177 and HisA181 in 1H33.
SoxX- Iron Porphyrine Interaction
In the crystal structure of 1H33 (B chain) the heme group is covalently
bound to the protein matrix through thioether bonds, between CysB42 Sγ
atom and MetB92 Sδ atom and the heme Cα positions 2 and 4, respectively
(Bamford et al., 2002). The Fe atom of the Iron porphyrine complex is
coordinated to the Nε atom of HisB46. Superimposition of the cytochrome
c motif of SoxX on the similar motif of 1H33 produced an r.m.s deviation
of 0.4 Å. In the SoxX-Iron porphyrine complex, Cys62, Met113 and His46
are found to be present at the identical positions.
Interaction of SoxA and SoxX
SoxA and SoxX are found to interact strongly with each other. The protein-protein
interface is found to contain mainly 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 side chains of the polar amino acid residues of SoxA and SoxX
are involved in H-bonding among themselves. The main chains of Gly196, Tyr213
and Ala253 of SoxA are found to form H-bond with the main chain of Ile123, Val80
and Ala64 of SoxX, respectively. The SoxAX complex is also stabilized by ionic
interactions. The residues of the proteins involved in the formation of ion-pair
are presented in Table 2. Out of the two cytochrome c motifs
of SoxA only the His206 of the second cytochrome c motif of SoxA and Ala64 and
His66 of the cytochrome c motif of SoxX are involved in the interaction. The
main chain of Ala64 of the cytochrome c motif of SoxX forms H-bond with the
side chain of Arg215 of SoxA. The side chain of His206 of SoxA is involved in
ionic interaction with that of His66.
Probable Mode of Action of the SoxAX Complex in Oxidation of Sulfur Anions
According to the mechanism of sulfur anion oxidation, the SoxAX complex
acquires electrons from the sulfur anions to facilitate the binding of sulfur
anions to the SoxY protein of the SoxYZ complex. From the structural arrangement
of SoxAX complex, it is easily notable that there is a highly positively charged
cluster of basic amino acid residues lining a channel, which provides access
to the anionic sulfur substrates. As observed in other proteins with well-conserved
cytochrome c motif (Bamford et al., 2002), SoxA too has an abundance
of positively charged amino residues on the surface of the SoxAX complex. The
catalytic channel is surrounded by the side chains of Arg215, Arg231, Asn233,
Arg243 and Arg249 as observed in other well-characterized cytochrome c proteins
(Bamford et al., 2002). From Table 1 it is evident
that, the residues of the second cytochrome c motif of SoxA as well as the cytochrome
c motif of SoxX are buried inside the SoxAX protein complex whereas the first
cytochrome c motif of SoxA is more accessible. Formation of SoxAX complex would
help to bring the two cytochrome c motifs (the second cytochrome c motif of
SoxA and the cytochrome c motif of SoxX) in close proximity (14 Å). This
closeness helps in the transport of electrons efficiently from one part of the
protein to the other part. According to the mechanism of sulfur anion oxidation
(Friedrich et al., 2001), the conserved cysteine residue at the N-terminal
region of SoxY protein binds the sulfur anion to form SoxY-thiocysteine-S-sulfate,
the first covalently bound sulfur adduct in the novel global sulfur anion oxidation
cycle. When the model of SoxY protein (pdb code: 2CVN) was docked to SoxAX complex,
it has been found that the N-terminal region of SoxY interacts strongly with
the positively charged amino acids residues (viz., Arg215, Arg231, Asn233, Arg243
and Arg249) lining the catalytic channel. Therefore, it may be concluded that
during the bio-geochemical sulfur oxidation reaction cycle, SoxAX complex first
binds the sulfur anions via the positively charged substrate-binding channel
on the surface of the SoxAX complex via polar interactions as found in case
of 1H33 (Bamford et al., 2002). Then SoxY interacts with the SoxAX
complex and removes the sulfur anion by making a covalent bond between the Sγ
atom of the N-terminal conserved cysteine residue of itself and the sulfur atom
of the sulfur anion. Since, the covalent bond strength is much more than polar
interactions (Lee, 1991), SoxY would easily remove the sulfur anion from SoxAX
complex to perform other steps of the reaction cycle.
Conclusions
In this study, we have tried to elucidate the structural basis of the involvements of SoxA and SoxX in the binding and in electron transport during oxidation of sulfur anions via the novel global sulfur oxidation cycle. We have described the three dimensional structures of the SoxA and SoxX proteins and established the geometry of the cytochrome c region and that of the active site of SoxAX complex. The docking studies with SoxA and SoxX have allowed us to identify the details of SoxA-SoxX interactions. The structural basis of the formation of the hetero-dimeric complex of SoxA-SoxX has also been demonstrated to predict the biochemical pathway of sulfur anion binding and transport of electrons via these proteins in the novel global sulfur cycle as SoxAX complex is the initiator of the sulfur anion oxidation pathway. Since there have been no previous reports regarding the structural biology of these proteins, results from this study will be important for the understanding of the three dimensional structures of SoxA and SoxX as well as to elucidate the structural basis of their mode of actions. Our model provides a rational framework for designing experiments aimed at determining the contribution of various amino acid residues in these proteins to predict the molecular basis of the interactions both among themselves as well as with various sulfur anions in these biologically as well as ecologically important micro-organisms.
Acknowledgements
We thank the anonymous referee for his or her valuable comments to improve the manuscript. This study was supported by grants from Department of Biotechnology, Government of India.