J. Cent. South Univ. Technol. (2007)04-0742-08
DOI: 10.1007/s11771-007-0141-x
Homology modeling and docking studies of
IscS from extremophile Acidithiobacillus ferrooxidans
LIU Yuan-dong(刘元东), DING Jian-nan(丁建南), QIU Guan-zhou(邱冠周), WANG Hai-dong(王海东)
(School of Resources Processing and Bioengineering, Central South University, Changsha 410083, China)
Abstract: The gene iscS-3 from Acidithiobacillus ferrooxidans may play a central role in the delivery of sulfur to a variety of metabolic pathways in this organism. For insight into the sulfur metabolic mechanism of the bacteria, an integral three-dimensional (3D) molecular structure of the protein encoded by this gene was built by homology modeling techniques, refined by molecular dynamics simulations, assessed by PROFILE-3D and PROSTAT programs and further used to search bind sites, carry out flexible docking with cofactor pyridoxal 5′-phosphate(PLP) and substrate cysteine and hereby detect its key residues. Through these procedures, the detail conformations of PLP-IscS(P-I) and cysteine-PLP-IscS(C-P-I) complexes were obtained. In P-I complex, the residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 have large interaction energies and/or hydrogen bonds fixation with PLP. In C-P-I complex, the amino group in cysteine is very near His106, Lys208 and PLP, the interaction energies for cysteine with them are very high. The above results are well consistent with those experimental facts of the homologues from other sources. Interestingly, the four residues of Glu105, Glu79, Ser203 and His180 in P-I docking and the residue of Lys213 in C-P-I docking also have great interaction energies, which are fitly conservation in IscSs from all kinds of sources but have not been identified before. From these results, this gene can be confirmed at 3D level to encode the iron-sulfur cluster assembly protein IscS and subsequently play a sulfur traffic role. Furthermore, the substrate cysteine can be presumed to be effectively recruited into the active site. Finally, the above detected key residues can be conjectured to be directly responsible for the bind and/or catalysis of PLP and cysteine.
Key words: bioleaching; IscS; Acidithiobacillus ferrooxidans; homology modeling; molecular dynamics; docking; pyridoxal 5’-phosphate(PLP); cysteine
1 Introduction
Acidithiobacillus ferrooxidans, a gram-negative and chemolithotrophic bacterium, is one of the most applied bacteria in industrial mineral processing to extract metals such as copper, lead, zinc, uranium, gold and nickel from their insoluble sulfide minerals[1]. The bioleaching environments for its survival are extreme acidity and toxicity[2]. One can wonder at how life is possible in such extreme environments. In fact, A. ferrooxidans can not only survive in those poisonous surroundings[3–4], but also take advantage of these conditions to gain energy for growth through oxidizing ferrous ion and reduced sulfur compounds as well as to convert the insoluble metal that sulfides to their soluble metal sulfates[5]. It is this capability of it that is utilized to extract metals from minerals. Therefore, understanding the mechanism of sulfur metabolism in A. ferrooxidans is of vital importance.
IscS is a cysteine desulfurase, which is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue[6]. Recent studies have revealed that a highly conserved gene cluster, iscSUA-hscBA-fdx, is essential for the general biogenesis of iron-sulfur proteins in bacteria[7]. IscS is a member of this gene cluster proteins, in which IscS functions mainly as sulfur donor for iron-sulfur cluster formation[8]. IscS is also able to transfer sulfur to ThiI, which is proposed to function as a sulfur transferase in thiamine and thionucleoside biosynthesis[9]. In addition, IscS may play a key role in the biosynthesis of biotin[10]. So IscS plays a central role in the delivery of sulfur to a variety of metabolic pathways. Several molecular structures of cysteine desulfurase from various sources that have been resolved by X-ray crystallography[11-12]. However, in all of those structures, a residues segment containing the active site cysteine and some residues in N-terminal and C-terminal regions cannot be directly resolved because these regions are disordered and have poor electron density. Moreover, none of them contains substrate cysteine.
It was reported that the genome sequence of A. ferrooxidans ATCC 23270 includes three genes, iscS-1, iscS-2 and iscS-3, which may encode the analogs to IscS. Among these genes, the iscS-1 and the iscS-2 are involved in the nitrogen fixation gene cluster, only the iscS-3 is in an iscSUA operon. So the gene iscS-3 may encode the general IscS and subsequently play a crucial role in the iron-sulfur cluster assembly for iron-sulfur proteins and other biological sulfur mobilization. But till now, there have been no theoretical efforts being made in the protein encoded by the iscS-3 and its three-dimensional (3D) structure remains to be elucidated. Moreover, it is an obstacle for directly obtaining the structure information of substrate-enzyme complex because substrate and enzyme react to provide product in a split second, whereas the structure resolution such as X-ray crystallography generally cost many hours. Knowledge of the detailed 3D structure and hereby the key residues identified are, however, essential for understanding its catalytic mechanism and function in the unique physiology of A. ferrooxidans.
In this study, with homology modeling techniques and molecular dynamics simulations, an integral 3D molecular structure of the protein encoded by gene iscS-3 from A. ferrooxidans (afIscS) was built, refined and assessed. The obtained structure was further used to search the bind sites, carry out flexible docking with cofactor PLP and substrate cysteine and hereby identify its key residues.
2 Computation and methods
The primary amino acidic sequence of the afIscS was retrieved from the A. ferrooxidans ATCC 23270 genome in the Institute for Genomic Research (TIGR). All computations were performed on the Dell Precision 470 workstation with Redhat Linux system using INSIGHT II software package developed by Accelrys Software Inc.
2.1 Three-dimensional model building
The HOMOLOGY module[13] in INSIGHT II was used to build the initial model of afIscS. Firstly, a sequence similarity search by BLAST program was carried out with each of the sequence separately against proteins whose solved structures had been deposited in the Protein Data Bank (PDB) to find related proteins as templates [14]. Then, MODELER program was performed to build the main 3D structure of afIscS. MODELER is an implementation of an automated approach to comparative modeling by satisfaction of spatial restraints[15]. For the remaining side chains, library values of ROTAMERS were adopted[16]. In order to construct the residues segment containing the active residue cysteine, a loop search algorithm over the databank of known crystal structure was used. The residues at the N-terminus and C-terminus were generated through END-REPAIR program. Through these procedures, an initial molecular structure model was completed.
The initial model was further improved through energy minimization (EM). After performing 800 steps of conjugate gradient (CG) minimization, molecular dynamics (MD) simulation was carried out to check their stability via performing 120 ps simulations at a constant temperature of 298 K. An explicit solvent model (TIP3P water)[17] was used, and the homology solvent model was constructed with a 2 nm water cap from the center of mass of afIscS. Finally, a conjugate gradient energy minimization of full protein was performed until the root mean square (RMS) gradient energy was lower than 42 GJ/(mol??m). All calculations mentioned above were accomplished by using DISCOVER_3 module in INSIGHT II. The consistent-valence force field (CVFF) was used for EM and MD simulations. In this step, the quality of the initial model was improved.
After the optimization procedure, the structure was assessed using PROFILE-3D and PROSTAT programs of INSIGHT II. The PROFILE-3D was used to measure the compatibility of an amino acid sequence with a known 3D protein structure[18]. The PROSTAT identified and listed the number of instances where structural features differ significantly from the average values calculated from known proteins[19].
2.2 Binding sites analysis
The BINDING-SITE module of INSIGHT II is a suite of programs for identifying and characterizing binding sites and functional residues from protein structure. In this study, the ACTIVESITE-SEARCH program was used to search the protein binding sites by locating cavities in the studied structure. Through comparing the conserved residues in family of the studied protein and combining the search results and other experiment results, the binding sites of afIscS were predicted. Those results were used to guide the following docking experiment.
2.3 Docking cofactor and substrate into binding pockets
The 3D structures of cofactor and substrate were built by BUILDER program and their geometries were further optimized through DISCOVER_3 program. For tracking the interacting modes among enzyme, cofactor and substrate, the automated molecular dockings among them were performed by docking program AFFINITY, which uses a combination of Monte Carlo type and stimulated annealing procedure to dock a gust molecule to a host one[20].
Firstly, the cofactor PLP was hosted into the binding pocket of afIscS apoenzyme under the guide of the above identified site. During the procedure, the best binding structure of the ligand to the receptor was automatically found by AFFINITY program based on the energy of the ligand and receptor complex. The potential function of the complex was assigned by using the CVFF, and the cell multipole approach was used for nonbonding interactions. To account for the solvent effect, the centered ligand-enzyme complex was solvated in a sphere of TIP3P water molecules with a radius of 3 nm. Finally, the docked complex of enzyme and cofactor was selected according to the criteria of interacting energy combined with geometrical matching quality.
Secondly, the substrate cysteine was docked into the active site of afIscS holoenzyme under the guide of the above identified site. Since PLP is covalently bonded with enzyme IscS in nature, the PLP bonded to enzyme was manually done and then the MD and EM processing was performed to remove the tensional structures before this docking. The docking procedure of cysteine was the same as these of PLP.
3 Results and discussion
3.1 Modeling structure of IscS from A. ferrooxidans
The amino acid sequence of the enzyme encoded by the iscS-3 gene from A. ferrooxidans ATCC 23270 was compared with each of the sequence of the known proteins in PDB by FASTA program. The results showed that a cysteine desulfurase from E. coli (PDB code 1P3W)[11] had the best sequence identity (65.71%) with this enzyme, so 1P3W was used to model the 3D structure of the protein. The sequence alignment between the target protein and the template protein is shown in Fig.1.
However, the structures of a residues segment containing the active site cysteine and some residues in N-terminal and C-terminal regions in 1P3W are disordered and cannot be directly resolved due to the low electron density in these regions. Moreover, the situations for all of the resolved structures in cysteine desulfurase are same to 1P3W. It makes an obstacle to further insight into the function of these enzymes. Fortunately, the homology modeling techniques of loop search algorithm can be used to settle such problems. Thus, Based on the structure of 1P3W, through the procedures of homology modeling, an initial integral structure of apo-afIscA was obtained.
The initial structure model was subsequently used to perform energy minimizations of 800 iterations and dynamics simulations of 120 ps. The variation of potential energy with time during the 120 ps of molecular dynamics simulations is plotted in Fig.2. Fig.2 shows that the potential energy falls rapidly in the first 50 ps, and then it decreases with very low deviation between two steps, and the dynamics simulations tend to equilibrium at 60 ps. Thus, the conformation at 120 ps was chosen as the final 3D structure for the further study. This model was further refined by EM optimization of 4 000 iterations, and then the final stable structure of afIscS was obtained after the root mean square (RMS) gradient energy was lower than 42 GJ/(mol??m).
Fig.1 Sequence alignment of afIscS with 1P3W
Fig.2 Variation of total potential energy during 120 ps of MD on afIscS (Total potential energy is averaged at 0.1 ps interval)
The final structure was further checked through PROFILE-3D and PROSTAT programs. The PROSTAT program was used to calculate the percent of backbone Φ–Ψ angles within the allowed Ramachandran region. The result is that 87.5% of the Φ–Ψ angles in the afIscS model lie in the core region of the Ramachandran plot. For the X-ray structure of 1P3W, the percent of backbone Φ–Ψ angles is 85.6%. PROSTAT program was also used to identify and list the number of instances where structural features differ significantly from average values in known proteins. The cutoff used for significant difference was six standard deviations from the reference values. The analytical results show that there are no significant differences between the calculated values for structure features in the modeled protein and the average values for those in known proteins for the total residues.
When checked by PROFILE-3D, the self- compatibility score for this protein is 181.95, which is higher than the low score(83.54) and close to the top score(185.65). This means that the structure model of afIscS is reasonable at the present level of theory. The checked detail results of PROFILE-3D are also presented in Fig.3. The compatibility scores above zero correspond to acceptable side chain environment, so Fig.3 indicates that all of the residues are reasonable. Both of the above results from PROFILE-3D and PROSTAT programs indicate that the modeled structure is reliable.
The monomer IscS from extremophile A. ferrooxidans ATCC 23270 consists of 407 amino acids and has a relative molecular mass of 45.4×103 by calculation. The modeled afIscS monomer can be subdivided into a small and a large domain (Fig.4). The small domain is composed of the N-terminal residues 1-19 and the C-terminal residues 267-407, which has 6 α helices, 5 β strands and 6 turns. The larger domain contains residues 20–266, which has 8 α helices, 7 β strands and 11 turns. The overall structure feature of this protein is typical for IscS protein. Moreover, the structures of N-terminal and C-terminal of this protein are random coils and disorders; the structure of the residues segment containing the active site cysteine randomly bulges out in the orientation of the large domain; these situations are also in line with the case that cysteine desulfurase is disordered in these regions.
Fig. 3 3D profiles verified results of afIscS model(residues with positive compatibility score are reasonably folded)
Fig. 4 Final 3D-structure and possible binding sites of afIscS (N-terminal and C-terminal of this protein are noted N and C respectively; the active residue cysteine is represented by balls and sticks; the possible binding sites are represented by filling their space with intersections and noted the number 1, 2 and 3, respectively)
3.2 Identification of binding regions of PLP and cysteine
In order to investigate the interaction among afIscS and cofactor PLP and substrate cysteine, the binding pocket is defined as a subset that contains residues in which any atoms are within 0.6 nm from PLP and cysteine. Three active sites are obtained using Binding-Site module in Insight II, the locations of the three sites in the 3D structure of afIscS are shown in Fig.4.
It is reported that the IscS proteins have a PLP-binding pocket and this pocket is involved in cofactor binding and substrate catalysis[11–12]. In this pocket, there exist two vital residues: a lysine, which performs the protonation of the ketimine intermediate before release of the product; a histidine, which performs most of the deprotonation and reprotonation steps during catalysis. There are also three conserved residues (a serine, a lysine and a histidine ), which play a very important role in anchoring the phosphate-group of PLP. Sequences alignment for IscSs from A. ferrooxidans and other sources (Fig.5) also show that there exist 5 highly conserved residues (Thr78, His106, Ser205, His207, and Lys208) in IscS proteins. Interestingly, all of the residues are located in the site 3.
The afIscS and 1P3W are well conserved in both sequence and structure, their biological functions should be identical. And thus the PLP binds can be presumed in a manner similar to both afIscS and 1P3W. Based on the experiment and the theoretical predicted results, in this study, site 3 is chosen as the more favorable binding site to dock the cofactor PLP and substrate cysteine. This site is composed of 17 residues (Gln14, Ala15, Ala77, Thr78, Thr81, His106, Ala108, Leu153, Asn157, Val184, Gln185, Ser205, His207, Lys208, Ser326, Tyr340, and Arg357).
3.3 Interaction between afIscS and PLP
PLP is an important cofactor in a wide range of biochemical reactions, including amino acid metabolism and antibiotic biosynthesis. All organisms must either produce PLP or acquire it through their diet. The 3D structure of PLP is made up of a pyridine ring connected by a -CHO group, a -OH group, a -CH3 group and a -CH2PO4 group.
The binding 3D conformation of the PLP-afIscS complex is described in Fig.6(a). This figure shows that the PLP is anchored in the center of the binding pocket.
Fig.5 Sequences alignment of IscSs from A. ferrooxidans and other sources(Only partial sequences are displayed; residues conserved in all sequences are marked with “*”; residues not conserved in all sequences but conserved in some sequences are marked with “:” or “.” based on the degree of conservation)
Fig.6 Docked 3D-structures of complex PLP-afIscS
(a) Overall structure; (b) Hydrogen bonds interaction between PLP and afIscS(Hydrogen bonds are represented by dash)
As is well known, hydrogen bonds play an important role in structure and function of the biological molecules, especially in enzyme catalysis and ligand binding. Six hydrogen bonds are formed between afIscS and PLP(Fig.6(b) and Table 1). Three hydrogen bonds are formed between phosphate oxygen atoms of PLP and the side-chains of Thr78, Ser205 and His207. Additional hydrogen bond interactions occur between the phenolate oxygen of PLP and Gln185, between the aldimine oxygen of PLP and Lys208, and between pyridine N1 of PLP and Asp182. These hydrogen bonding interactions enhance the stability of the ligand-enzyme complex.
Table 1 Hydrogen bonds between PLP and bind site residues of afIscS
To determine the key residues that comprise the binding pocket of the model, the interaction energy of PLP with each individual amino acid in the enzyme was also calculated. Significant binding-site residues in the models were identified by the total interaction energy between the cofactor and each amino acid residue in the afIscS. The relative importance of each residue can be inferred by its rank order of interaction energy. This kind of identification, compared with a definition based on the distance from the substrate, can clearly show the relative significance for every residue.
Table 2 lists the interaction energies for PLP-afIscS complex. It is evident from Table 2 that the PLP-afIscS complex has a favorable total interaction energy, the electrostatic energy, the van der Waals energy and the total interaction energy for the total residues are -61.236, -42.600 and -103.836 kJ/mol, respectively. These results indicate that the attractive interaction is important and in this case both the van der Waals and electrostatic energies are important for determining the binding orientations.
The total interaction energy, Etotal, between the PLP and Lys208 is -24.646 kJ/mol and in which the primary interaction energy is electronic interaction one (Eele=-43.604 kJ/mol). The residues of Ser205, Glu105, Glu79, and His207 have the similar behavior as Lys208 and the interaction energies of these residues with PLP are mainly contributed to electronic interaction. But, for the residue of His106, Ser203 and His180 the interaction energy with PLP is mainly contributed to van der Waals interaction. For the residues of Thr78, Asp182 and Gln185, the interaction energies with PLP are equal in the gross by van der Waals and electronic interaction.
Table 2 Total energy (Etotal), electrostatic energy (Eele), and van-der-Waals energy (Evdw) between PLP and individual residues (|Etotal| >1.500 kJ/mol listed in energy rank order)
Through the interaction analysis, the total interaction energies for the residues of Lys208, His106, Thr78, Asp182, Gln185, Ser205, Glu105, Glu79, His207, Ser203 and His180 are prominent compared with the other residues, so these residues can be conjectured to be important anchoring residues for PLP and have main contribution to the cofactor interaction. Previous experimental results have demonstrated that the IscS from E. coli has 7 vital residues[11]: Lys206, which anchors the PLP group in the active site by formation of an internal aldimine Schiff base; His104, which functions as an acid–base catalyst in several protonation or deprotonation steps during catalysis; Thr76, Ser203 and His205, which play a very important role in anchoring the phosphate group of PLP; Asp180 and Gln183, which play an important role in fixing the ring of PLP. A NifS-like cysteine desulfurase protein from Thermotoga maritime[12] also exist residue counterparts to IscS from E.coli. The residues homologous to Lys206, His104, Thr76, Ser203, His205, Asp180 and Gln183 of IscS from E.coli are Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 of afIscS, respectively. Obviously, the results of the 7 residues are well consistent with those experimental facts[11–12] based on the total interacting energy and/or hydrogen bonds fixation. However, the four residues of Glu105, Glu79, Ser203 and His180 are new identified important residues according to the interaction energy, which are fitly conservation in IscSs from all kinds of sources (Fig.5) but have not been identified before.
Additionally, before formation of the Michaelis complex between holoenzyme afIscS and substrate cysteine, the PLP is covalently bonded to a lysine[11–12]. The above docking results are also supported the conclusion because the –CHO group in PLP is very close to the residue Lys208 (Fig.6(b)) and the interaction energy between them is very high (Table 1).
3.4 Interaction between holoenzyme afIscS and substrate cysteine
Cysteine is one of the 20 necessary amino acids for protein biosynthesis in all organisms. During the initial steps of substrate reaction (Fig.7(a)), the cysteine enters the active pocket and forms the Michaelis complex, the substrate amino group is deprotonated by His106 and then nucleophilicly attacks on C4A atom in PLP to form the external aldimine. The substrate docking in this paper mainly aimed at the formation of Michaelis complex. The results indicate that the amino group in cysteine is near to His106 and PLP and the distances between the HN2 atom in amino group of cysteine and the NE2 atom in His106, and between the HN3 atom in amino group of cysteine and the C4A atom in PLP are 0.181 nm and 0.242 nm (Fig.7(b)), respectively, which are in line with the initial process of substrate reaction mechanism(Fig.7(a)).
Table 3 lists the interaction energy information of cysteine with the afIscS holoenzyme. The cysteine-PLP-afIscS complex has a favorable total interaction energy, the electrostatic energy, the van der Waals energy and the total interaction energy for the total residues are -40.815, -30.840 and -71.655 kJ/mol, respectively. The interaction energy corresponds only to the enthalpic contribution to the free energy of binding. The results indicate that in this case both the van der Waals and electrostatic energies are important for determining the substrate binding orientation, and the large attractive interaction energy is helpful for effectively recruiting cysteine. The total interaction energy, Etotal, between the cysteine and His106 is -27.711 kJ/mol and in which the primary interaction energy is electronic energy (Eele -17.841 kJ/mol). The residues of Lys208, Lys213 and cofactor PLP have the similar behavior as His106 and the interaction energies of these residues with cysteine are mainly contributed to electronic interaction. Interestingly, the total interaction of His106, Lys208, Lys213 and cofactor PLP are prominent compared with the other residues. These results indicate that His106, Lys208, Lys213 and cofactor PLP are important active components for cysteine and have main contribution to the substrate interaction. Obviously, the results of the cofactor PLP and the two residues of His106 and Lys208 are well consistent with those experimental facts[11–12]. However, Lys213 is a new identified important residue according to the interaction energy, which is also fitly conservation in IscSs from all kinds of sources (Fig.5) but has not been identified before.
Fig.7 Interaction between substrate cysteine and afIscS
(a) Schematic of initial steps for possible reaction mechanism of afIscS; (b) Position of substrate docking
Table 3 Total energy (Etotal), electrostatic energy (Eele), and van-der-Waals energy (Evdw) between substrate cysteine and individual residues (|Etotal|>1. 750 kJ/mol listed in energy rank order)
4 Conclusions
1) The integral 3D molecular model of the enzyme encoded by gene iscS-3 from extremophile A. ferrooxidans is built and refined. The evaluation results by PROFILE-3D and PROSTAT programs indicate that this model is reliable. From the information of modeled structure and interactions with PLP and cysteine, this gene can be confirmed at 3D level to encode the IscS and subsequently play a sulfur traffic role in organism of the bacteria.
2) According to the docking with cofactor PLP, the PLP can be firmly anchored in the binding pocket of IscS from A. ferrooxidans. The residues of Lys208, His106, Thr78, Ser205, His207, Asp182 and Gln185 are directly responsible for PLP binding and catalytic function in IscS from A. ferrooxidans.
3) According to the docking with substrate cysteine, the cysteine can be effectively recruited into the active site of IscS from A. ferrooxidans and subsequently activated. The residues of Lys208, His106 and Lys213 are of vital importance in the catalysis of cysteine .
4) The detailed 3D structure, interaction information and the key residues identified are helpful for guiding the site-directed mutagenesis investigation and understanding the catalytic mechanism of the enzyme and subsequently insight into the sulfur metabolism of the A. ferrooxidans so as to finally serve for industrial bioleaching.
References
[1] RAWLINGS D E, KUSANO T. Molecular genetics of Thiobacillus ferrooxidans[J]. Microbiological Reviews, 1994, 58(1): 39-55.
[2] BAKER B J, BANFIELD J F. Microbial communities in acid mine drainage[J]. FEMS Microbiology Ecology, 2003, 44(2): 139-152.
[3] TUOVINEN O H, NIEMELA S I, GYLLENBERG H G. Tolerance of Thiobacillus ferrooxidations to some metals[J]. Antonie Van Leeuwenhoek, 1971, 37(4): 489–496.
[4] BANERJEE P C. Genetics of metal resistance in acidophilic prokaryotes of acidic mine environments[J]. Indian Journal of Experimental Biology, 2004, 42(1): 9–25.
[5] QUATRINI R, APPIA-AYME C, DENIS Y, et al. Insights into the iron and sulfur energetic metabolism of Acidithiobacillus ferrooxidans by microarray transcriptome profiling[J]. Hydrometallurgy,2006, 83(1/4):263–272.
[6] MIHARA H, ESAKI N. Bacterial cysteine desulfurases: Their function and mechanisms[J]. Applied Microbiology and Biotechnology, 2002, 60(1/2): 12–23.
[7] JOHNSON D C, DEAN D R, SMITH A D, et al. Structure, function, and formation of biological iron-sulfur clusters[J]. Annual Review of Biochemistry, 2005, 74: 247–281.
[8] URBINA H D, SILBERG J J, HOFF K G, et al. Transfer of sulfur from IscS to IscU during Fe-S cluster assembly[J]. Journal of Biological Chemistry, 2001, 276(48): 44521-44526.
[9] LAUHON C T. Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli[J]. Journal of Bacteriology, 2002, 184(24): 6820–6829.
[10] BUI B T S, ESCALETTES B, CHOTTARD F, et al. Enzyme-mediated sulfide production for the reconstitution of [2Fe–2S] clusters into apo-biotin synthase of Escherichia coli[J]. European Journal of Biochemistry, 2000, 267(9): 2688–2694.
[11] CUPP-VICKERY J R, URBINA H, VICKERY L E. Crystal structure of IscS-A cysteine desulfurase from Escherichia coli[J]. Journal of Molecular Biology, 2003, 330(5): 1049–1059.
[12] KAISER J T, CLAUSEN T, BOURENKOW G P, et al. Crystal structure of a NifS-like protein from Thermotoga maritima: Implications for iron sulphur cluster assembly[J]. Journal of Molecular Biology,2000, 297(2): 451-464.
[13] BLUNDELL T L, SIBANDA B L, STERNBERG M J E, et al. Knowledge-based prediction of protein structures and the design of novel molecules[J]. Nature, 1987, 326(6111): 347–352.
[14] LIPMAN D J, PEARSON W R. Rapid and sensitive protein similarity searches[J]. Science, 1985, 227(4693):1435–1441.
[15] SALI A, POTTERTON L, YUAN F, et al. Evaluation of comparative protein modeling by MODELLER[J]. Proteins, 1995, 23(3): 318–326.
[16] PONDER J W, RICHARDS F M. Tertiary templates for proteins: Use of packing criteria in the enumeration of allowed sequences for different structural classes[J]. Journal of Molecular Biology,1987, 193(4): 775–791.
[17] JORGENSEN W L, CHANDRASEKHAR J, MADURA J D, et al. Comparison of simple potential functions for simulating liquid water[J]. Journal of Chemical Physics, 1983, 79(2): 926–935.
[18] LUTHY R, BOWIE J U, EISENBERG D. Assessment of protein models with three-dimensional profiles[J]. Nature, 1992, 356(6364): 83–85.
[19] MORRIS A L, MACARTHUR M W, HUTCHINSON E G, et al. Stereochemical quality of protein structure coordinates[J]. Proteins, 1992, 12(4): 345–364.
[20] KUNTZ I D, BLANEY J M, OATLEY S J, et al. A geometric approach to macromolecule-ligand interactions[J]. Journal of Molecular Biology,1982, 161(2): 269–288.
(Edited by YANG Hua)
Foundation item: Project(2004CB619201) supported by the National Basic Research Program of China; Project(50321402) supported by the National Natural Science Foundation of China
Received date: 2007-03-02; Accepted date: 2007-04-18
Corresponding author: QIU Guan-zhou, Professor, PhD; Tel: +86-731-8879815; E-mail: lydcsu@yahoo.com.cn