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1.
Kuo-Liang Su Ya-Fan Liao Hui-Chih Hung Guang-Yaw Liu 《The Journal of biological chemistry》2009,284(39):26768-26777
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1–3). Ornithine decarboxylase (ODC,3 EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8–15). They also have been associated with human diseases and a variety of cancers (16–26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6, 11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity requires the formation of a dimer (29–31). X-ray structures of the ODC enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in PLP interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate (27, 32–41).ODC undergoes a unique ubiquitin-independent proteasomal degradation via a direct interaction with the regulatory protein antizyme (AZ). Binding of AZ promotes the dissociation of the ODC homodimers and targets ODC for degradation by the 26 S proteasome (42–46). Current models of antizyme function indicate that increased polyamine levels promote the fidelity of the AZ mRNA translational frameshift, leading to increased concentrations of AZ (47). The AZ monomer selectively binds to dimeric ODC, thereby inactivating ODC by forming inactive AZ-ODC heterodimers (44, 48–50). AZ acts as a regulator of polyamine metabolism that inhibits ODC activity and polyamine transport, thus restricting polyamine levels (4, 5, 51, 52). When antizymes are overexpressed, they inhibit ODC and promote ubiquitin-independent proteolytic degradation of ODC. Because elevated ODC activity is associated with most forms of human malignancies (1), it has been suggested that antizymes may function as tumor suppressors.In contrast to the extensive studies on the oncogene ODC, the endogenous antizyme inhibitor (AZI) is less well understood. AZI is homologous to the enzyme ODC. It is a 448-amino acid protein with a molecular mass of 50 kDa. However, despite the homology between these proteins, AZI does not possess any decarboxylase activity. It binds to antizyme more tightly than does ODC and releases ODC from the ODC-antizyme complex (53, 54). Both the AZI and AZ proteins display rapid ubiquitin-dependent turnover within a few minutes to 1 h in vivo (5). However, AZ binding actually stabilizes AZI by inhibiting its ubiquitination (55).AZI, which inactivates all members of the AZ family (53, 56), restores ODC activity (54), and prevents the proteolytic degradation of ODC, may play a role in tumor progression. It has been reported that down-regulation of AZI is associated with the inhibition of cell proliferation and reduced ODC activity, presumably through the modulation of AZ function (57). Moreover, overexpression of AZI has been shown to increase cell proliferation and promote cell transformation (58–60). Furthermore, AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function (60, 61). These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis.It is now known that ODC exists as a dimer and that AZI may exist as a monomer physiologically (62). Fig. 1 shows the dimeric structures of ODC (Fig. 1A) and AZI (Fig. 1B). Although structural studies indicate that both ODC and AZI crystallize as dimers, the dimeric AZI structure has fewer interactions at the dimer interface, a smaller buried surface area, and a lack of symmetry of the interactions between residues from the two monomers, suggesting that the AZI dimer may be nonphysiological (62). In this study, we identify the critical amino acid residues governing the difference in dimer formation between ODC and AZI. Our preliminary studies using analytical ultracentrifugation indicated that ODC exists as a dimer, whereas AZI exists in a concentration-dependent monomer-dimer equilibrium. Multiple sequence alignments of ODC and AZI from various species have shown that residues 277, 331, 332, and 389 are not conserved between ODC and AZI (Open in a separate windowFIGURE 1.Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).
Open in a separate window 相似文献
TABLE 1
Amino acid residues at the dimer interface of human ODC and AZIHuman ODC | Residue | Human AZI |
---|---|---|
Nonconserved | ||
Arg | 277 | Ser |
Tyr | 331 | Ser |
Asp | 332 | Glu |
Tyr | 389 | Asp |
Conserved | ||
Asp | 134 | Asp |
Lys | 169 | Lys |
Lys | 294 | Lys |
Tyr | 323 | Tyr |
Asp | 364 | Asp |
Gly | 387 | Gly |
Phe | 397 | Phe |
2.
3.
Ezgi Karaca Adrien S. J. Melquiond Sjoerd J. de Vries Panagiotis L. Kastritis Alexandre M. J. J. Bonvin 《Molecular & cellular proteomics : MCP》2010,9(8):1784-1794
Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of experimental information (e.g. from mass spectrometry, nuclear magnetic resonance, cryoelectron microscopy, etc.) will remain highly desirable to obtain reliable results.Proteins are the wheels and millstones of the complex machinery that underlies human life. Catalyzing a huge diversity of chemical processes, proteins work in close association with other biomolecules: nucleic acids, sugars, lipids, and other proteins. This huge network of protein interactions enables the cell to respond quickly to changes in the environment, such as temperature, oxygen, or nutrient concentration. However, to fully understand this network, insights at the atomic level are needed.In the wake of the elucidation of the human genome (1, 2), many structural genomics projects are solving the structures of what is now becoming a considerable fraction of the human proteome (3). These projects are now moving to the next level, which is solving the atomic resolution structures of protein complexes. However, this is a challenge that is considerably greater than obtaining the structures of single proteins. First of all, a protein can take part in 10 interactions on average; thus, the number of complexes is expected to be at least an order of magnitude larger than the proteome, and their composition can even vary over time. Second, associations between subunits in protein complexes are often weak and reversible, which make purification and crystallization difficult. Finally, there are some very well studied classes of interactions, such as enzyme-inhibitor, antibody-antigen, and GTPase-GAP (GTPase-activating protein) interactions, but these classes represent binary interactions between proteins. In contrast, many of the most important functions in the cell are carried out by large, dynamic molecular assemblies, such as the ribosome, the proteasome, the spliceosome, RNA polymerases, and the nuclear pore complex (4, 5). For such assemblies, high resolution methods such as x-ray crystallography and NMR spectroscopy often provide atomic level information at the level of individual subunits or subcomplexes, but they typically encounter difficulties at the level of the full complex.Fortunately, low resolution information about protein complexes can often be obtained. Affinity purification (6, 7) followed by mass spectrometry is a high throughput technique to study the composition of a complex. However, dissociation inside the mass spectrometer can be a problem for transient or unstable complexes in which case chemical cross-linking can help. Once the composition of the complex is known, there is a variety of experimental techniques available to obtain structural information on the complex. The most detailed information can be gathered by using data obtained from various NMR experiments, for example chemical shift perturbations (8) or residual dipolar couplings (9); unfortunately, NMR is limited to complexes that are fairly small in size, making its applicability in the context of large assemblies less suited. Techniques that provide information about the shape of a protein complex, such as small angle x-ray scattering (SAXS),1 cryoelectron tomography, and single molecule cryoelectron microscopy (cryo-EM), are more suited to characterize large complexes. Unfortunately, all of these techniques suffer from limitations in resolution that are either fundamental or caused by structural heterogeneities of the complex.A well known approach to obtain information on residues at an interface is site-directed mutagenesis (10). In principle, a loss of binding affinity indicates that the mutated residue mediates the interaction, although the reverse is not true. Also, one must take care of secondary effects, such as unfolding or conformational change caused by the mutation. Apart from that, very detailed information about interface residues can be obtained by extensive mutagenesis experiments, such as alanine scanning and double mutant cycles. Mass spectrometry offers the opportunity to get peptide level or residue level information about protein interfaces by accurate mass measurements of peptides from the protein complex, generated either a priori through proteolytic cleavage, or inside the mass spectrometer (MS/MS). For example, interface residues can be identified as residues that undergo slower hydrogen/deuterium exchange upon complex formation. This process can be monitored at the peptide level by mass spectrometry (or in smaller complexes, at the residue level by NMR), although this method is very sensitive to noise caused by conformational changes upon binding. In the same way, radical probe MS (RP-MS) uses differences in oxidation of residues by hydroxyl radicals generated in the mass spectrometer to identify interface residues. Finally, chemical cross-linking followed by MS can provide direct information about residue contact sites between different binding partners of the complex. Several cross-linking reagents can provide complementary information. However, it has been reported that the cross-linkers may disrupt the structure of the protein complex and that care should therefore be taken to interpret the results (11).There is a need for computational approaches to translate this low resolution information into atomic resolution models that can provide functional and mechanistic insights. One of the most promising approaches is docking, the prediction of the structure of a complex starting from the free, unbound structures of its constituents. In recent years, docking methods have made much progress in the blind prediction of the structure of protein complexes as seen in the recent rounds of the critical assessment of prediction of interactions (CAPRI) experiment (12, 13). Most docking methods are ab initio, which means that experimental data are not required. However, it is possible in several ab initio methods to use experimentally determined interface residues in the docking: in MolFit (14, 15) and ATTRACT (16, 17), it is possible to up-weight the interaction scores of interface residues; in ZDOCK (18, 19), it is possible to block non-interface residues; and in PatchDock (20, 21), ZDOCK, pyDock (22, 23), and several other methods, it is possible to filter the docking results based on experimental information. Next to purely ab initio approaches, there are also methods that make use of different types experimental information, for example PROXIMO (24), based on RP-MS data, and MultiFit (25), a hybrid fitting/docking approach based on electron microscopy data.A method that distinguishes itself from the variety of above mentioned docking approaches is HADDOCK (26–28). In HADDOCK, the docking can be driven by a variety of experimental data using information about interface, contacts, and relative orientations inside a complex simultaneously. Originally developed for NMR data, HADDOCK is able to deal with a large variety of experimental data as shown in 26–28) and “Materials and Methods” for more details.) HADDOCK has performed very well in translating these data into structures and structural models. More than 60 Protein Data Bank structures calculated using HADDOCK have been deposited to date as experimental structures in the Protein Data Bank (29). Moreover, HADDOCK has shown a strong performance in CAPRI. Finally, HADDOCK is a general purpose program that can integrate many kinds of data, but even with a single source of data it is able to perform as well as more specialized programs: for example, HADDOCK was able to closely reproduce the NMR-calculated E2A-HPr complex using only chemical shift perturbation data. For the ribonuclease S-protein-peptide complex (Protein Data Bank code 1J80 (30)) for which RP-MS data are available, PROXIMO was able to closely reproduce the crystal structure (root mean square deviation (r.m.s.d.) of the top scoring model from the reference crystal structure is 1.26 Å); using the same data, HADDOCK could get even closer with an r.m.s.d. of only 0.68 Å from the crystal structure (results not shown).
Open in a separate windowa Nuclear Overhauser effects.Most docking methods are designed to deal with just two molecules, making their application limited with regard to large macromolecular assemblies. In most programs, multicomponent complexes can be assembled by adding each component one at a time, whereas simultaneous docking of the whole complex is typically not possible. Recently five ab initio docking programs (MolFit (31, 32), ClusPro (33), Rosetta (34), M-ZDOCK (35), and SymmDock (36)) gave birth to specific versions for the prediction of the symmetric multimers. Among these programs, MolFit, ClusPro, and Rosetta perform a rotational/translational search about the proper symmetry axes. These programs can deal with different types of cyclic and dihedral symmetries. Different than the other two, Rosetta is able to assemble complexes having helical and icosahedral symmetries. M-ZDOCK and SymmDock are suited for the prediction of macromolecules with cyclic symmetries. However, the ability to deal with arbitrary large molecular assemblies is currently rare. CombDock (37), which was developed by the team of SymmDock, can build hetero-oligomer complexes, but it does not have a symmetry option. Only HADDOCK can deal with molecular complexes that are hetero-oligomers or homo-oligomers with arbitrary symmetry operators between and within each component.The flexibility of HADDOCK comes at a price: it requires the user to have the structure calculation program CNS (38) installed and a considerable degree of expertise in its usage and molecular modeling in general, and it requires a cluster of computers. To alleviate this problem and to open up HADDOCK for a wide community, we have recently developed the HADDOCK web server (27). The server offers multiple web interfaces, ranging from very simple and user-friendly to very powerful and flexible, exposing the full range of HADDOCK options to the expert user. However, up until now, the HADDOCK server was unable to deal with more than two molecules. Here we present a novel web interface for multibody docking of complexes. Like the HADDOCK program itself, the server supports the docking of up to six molecules simultaneously; all HADDOCK options, including symmetry restraints, are made available to the user. Even larger assemblies can in principle be modeled if the docking is performed in an incremental way. Here we demonstrate the performance of the multibody server on a small benchmark comprising complexes of various symmetries and increasing numbers of components (from three to five). To drive the docking, bioinformatics interface predictions and/or available experimental information were used. The HADDOCK server is available on line. http://haddock.chem.uu.nl. 相似文献
Table I
Various experimental data that can be incorporated into HADDOCKExperimental data | HADDOCK representation |
---|---|
Mutagenesis data | Active and passive residues |
Hydrogen/deuterium exchange data | Active and passive residues |
Bioinformatics interface predictions | Active and passive residues |
Mass spectrometry data | |
Cross-linking data | Custom CNS restraints |
Radical probe mass spectrometry | Active and passive residues |
Limited proteolysis mass spectrometry | Active and passive residues or directly as an MTMDAT-generated HADDOCK parameter file |
NMR data | |
Chemical shift perturbation data | Active and passive residues |
Cross-saturation experiments | Active and passive residues |
Residual dipolar couplings | Directly |
Diffusion anisotropy restraints | Directly |
NOEsa as custom CNS restraints | Custom CNS restraints |
Dihedral angles | Directly |
Hydrogen bonds | Directly |
Paramagnetic restraints | Under development |
Shape data | |
SAXS | Under development |
EM | Under development |
4.
5.
Denise W?tzlich Markus J. Br?cker Frank Uliczka Markus Ribbe Simone Virus Dieter Jahn Jürgen Moser 《The Journal of biological chemistry》2009,284(23):15530-15540
Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL2 or ChlL2 of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)2 or (ChlNB)2. Analogously, subunit BchX2 of the COR enzymes delivers electrons to subunit (BchYZ)2. Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)2 and BchL2 from Chlorobaculum tepidum or (ChlNB)2 and ChlL2 from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL2 with both subunits, ChlN and ChlB, of the (ChlNB)2 tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr127 of subunit ChlL, Leu70 and Val107 of subunit ChlN, and Gly66 of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL2 or ChlL2 part of DPOR was exchanged with electron-transferring BchX2 subunits of COR and NifH2 of nitrogenase. Active chimeric DPOR was generated via a combination of BchX2 from C. tepidum or Roseobacter denitrificans with (BchNB)2 from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH2 from Azotobacter vinelandii in combination with (BchNB)2 from C. tepidum or (ChlNB)2 from P. marinus and T. elongatus, respectively.Chlorophyll and bacteriochlorophyll biosynthesis, as well as nitrogen fixation, are essential biochemical processes developed early in the evolution of life (1). During biological fixation of nitrogen, nitrogenase catalyzes the reduction of atmospheric dinitrogen to ammonia (2). Enzyme systems homologous to nitrogenase play a crucial role in the formation of the chlorin and bacteriochlorin ring system of chlorophylls (Chl)2 and bacteriochlorophylls (Bchl) (3, 4) (Fig. 1a). For the synthesis of both Chl and Bchl, the stereospecific reduction of the C-17-C-18 double bond of ring D of protochlorophyllide (Pchlide) catalyzed by the nitrogenase-like enzyme light-independent (dark-operative) protochlorophyllide oxidoreductase (DPOR) results in the formation of chlorophyllide (Chlide) (Fig. 1a, left) (5, 6). DPOR enzymes consist of three protein subunits which are designated BchN, BchB and BchL in Bchl-synthesizing organisms and ChlN, ChlB and ChlL in Chl-synthesizing organisms. A second reduction step at ring B (C-7-C-8) unique to the synthesis of Bchl converts the chlorin Chlide into a bacteriochlorin ring structure to form bacteriochlorophyllide (Bchlide) (Fig. 1a, right, Bchlide). This reaction is catalyzed by another nitrogenase-like enzyme, termed chlorophyllide oxidoreductase (COR) (7). COR enzymes are composed of subunits BchY, BchZ, and BchX.Open in a separate windowFIGURE 1.Comparison of the three subunit enzymes DPOR, COR, and nitrogenase. a, during Chl and Bchl biosynthesis, ring D is stereospecifically reduced by the nitrogenase-like enzyme DPOR (subunit composition BchL2/(BchNB)2 or ChlL2/(ChlNB)2) leading to the chlorin Chlide. Subunits N, B, and L are named ChlN, ChlB, and ChlL in Chl-synthesizing organisms and BchN, BchB, and BchL in Bchl-synthesizing organisms. The synthesis of Bchl additionally requires the stereospecific B ring reduction by a second nitrogenase-like enzyme called COR, with the subunit composition BchX2/(BchYZ)2. COR catalyzes the formation of the bacteriochlorin Bchlide. Subunits Y, Z, and X of the COR enzyme are named BchY, BchZ, and BchX. b, the homologous nitrogenase complex has the subunit composition NifH2/(NifD/NifK)2. Rings A–E and the carbon atoms are designated according to IUPAC nomenclature (41). R is either a vinyl or an ethyl moiety. The position marked by an asterisk indicates either a vinyl or a hydroxyethyl moiety (42).All subunits share significant amino acid sequence homology to the corresponding subunits of nitrogenase, which are designated NifD, NifK, and NifH, respectively (1) (compare Fig. 1, a and b). Whereas subunits BchL or ChlL, BchX and NifH exhibit a sequence identity at the amino acid level of ∼33%, subunits BchN or ChlN, BchY, NifD, and BchB or ChlB, BchZ, and NifK, respectively, show lower sequence identities of ∼15% (1). For all enzymes a common oligomeric protein architecture has been proposed consisting of the heterotetrameric complexes (BchNB)2 or (ChlNB)2, (BchYZ)2, and (NifD/NifK)2, which are completed by a homodimeric protein subunit BchL2 or ChlL2, BchX2, and NifH2, respectively (compare Fig. 1, a and b) (3, 7, 8).Nitrogenase is a well characterized protein complex that catalyzes the reduction of nitrogen to ammonia in a reaction that requires at least 16 molecules of MgATP (2, 9, 10). During nitrogenase catalysis, subunit NifH2 (Fe protein) associates with and dissociates from the (NifD/NifK)2 complex (MoFe protein). Binding, hydrolysis of MgATP and structural rearrangements are coupled to sequential intersubunit electron transfer. For this purpose, NifH2 contains an ATP-binding motif and an intersubunit [4Fe-4S] cluster coordinated by two cysteine residues from each NifH monomer (1, 11). Electrons from this [4Fe-4S] cluster are transferred via a [8Fe-7S] cluster (P-cluster) onto the [1Mo-7Fe-9S-X-homocitrate] cluster (MoFe cofactor). Both of the latter clusters are located on (NifD/NifK)2, where dinitrogen is reduced to ammonia (10). Three-dimensional structures of NifH2 in complex with (NifD/NifK)2 revealed a detailed picture of the dynamic interaction of both subcomplexes (8, 12).Based on biochemical and bioinformatic approaches, it has been proposed that the initial steps of DPOR reaction strongly resemble nitrogenase catalysis. Key amino acid residues essential for DPOR function have been identified by mutagenesis of the enzyme from Chlorobaculum tepidum (formerly denoted as Chlorobium tepidum) (3). The catalytic mechanism of DPOR includes the electron transfer from a “plant-type” [2Fe-2S] ferredoxin onto the dimeric DPOR subunit, BchL2, carrying an intersubunit [4Fe-4S] redox center coordinated by Cys97 and Cys131 in C. tepidum. Analogous to nitrogenase, Lys10 in the phosphate-binding loop (P-loop) and Leu126 in the switch II region of DPOR were found essential for DPOR catalysis. Moreover, it was shown that the BchL2 protein from C. tepidum does not form a stable complex with the catalytic (BchNB)2 subcomplex. Therefore, a transient interaction responsible for the electron transfer onto protein subunit (BchNB)2 has been proposed (3).The subsequent [Fe-S] cluster-dependent catalysis and the specific substrate recognition at the active site located on subunit (BchNB)2 are unrelated to nitrogenase. The (BchNB)2 subcomplex was shown to carry a second [4Fe-4S] cluster, which was proposed to be ligated by Cys21, Cys46, and Cys103 of the BchN subunit and Cys94 of subunit BchB (C. tepidum numbering) (3). No evidence for any type of additional cofactor was obtained from biochemical and EPR spectroscopic analyses (5, 13). Thus, despite the same common oligomeric architecture, the catalytic subunits (BchNB)2 and (ChlNB)2 clearly differ from the corresponding nitrogenase complex, as no molybdenum-containing cofactor or P-cluster equivalent is employed (5, 14). From these results it was concluded that electrons from the [4Fe-4S] cluster of (BchNB)2 or (ChlNB)2 are transferred directly onto the Pchlide substrate at the active site of DPOR.The second nitrogenase-like enzyme, COR, catalyzes the reduction of ring B of Chlide during the biosynthesis of Bchl (7). Therefore, an accurate discrimination of the ring systems of the individual substrates is required. COR subunits share an overall amino acid sequence identity of 15–22% for BchY and BchZ and 31–35% for subunit BchX when compared with the corresponding DPOR subunits (supplemental Figures S2–S4). In amino acid sequence alignments of BchX proteins with the closely related BchL or ChlL subunits of DPOR, both cysteinyl ligands responsible for [4Fe-4S] cluster formation and residues for ATP binding are conserved (1). Furthermore, all cysteinyl residues characterized as ligands for a catalytic [4Fe-4S] cluster in (BchNB)2 or (ChlNB)2 are conserved in the sequences of subunits BchY and BchZ of COR (7). These findings correspond to a recent EPR study in which a characteristic signal for a [4Fe-4S] cluster was obtained for the COR subunit BchX2 as well as for subunit (BchYZ)2 (15). These results indicate that the catalytic mechanism of COR strongly resembles DPOR catalysis. In vitro assays for nitrogenase as well as for DPOR and COR make use of the artificial electron donor dithionite in the presence of high concentrations of ATP (7, 16, 17).
Open in a separate windowIn this study, we investigated the transient interaction of the dimeric subunit BchL2 or ChlL2 with the heterotetrameric (BchNB)2 or (ChlNB)2 complex, which is essential for DPOR catalysis. We make use of the individually purified DPOR subunits BchL2 and (BchNB)2 from the green sulfur bacterium C. tepidum and ChlL2 and (ChlNB)2 from the prochlorophyte Prochlorococcus marinus and from the cyanobacterium Thermosynechococcus elongatus. The individual combination of (BchNB)2 or (ChlNB)2 complexes and BchL2 or ChlL2 proteins from these organisms resulted in catalytically active chimeras of DPOR. These results enabled us to propose conserved regions of the postulated docking surface, which were subsequently verified in a mutagenesis study. To elucidate the potential evolution of the electron-transferring subunit of nitrogenase and nitrogenase-like enzymes, we also analyzed chimeric enzymes consisting of DPOR subunits (BchNB)2 or (ChlNB)2 in combination with subunits BchX2 from C. tepidum and R. denitrificans of the COR enzyme and with subunit NifH2 of nitrogenase from Azotobacter vinelandii, respectively. 相似文献
TABLE 1
Amino acid sequence identities of the individual subunits of DPOR, COR, and nitrogenaseAmino acid sequences of the individual subunits of DPOR, COR, and nitrogenase employed in the present study (compareFig. 3A) were aligned by using the ClustalW method in MegAlign (DNASTAR), and sequence identities were calculated.DPOR | COR | Nitrogenase | |||||||
---|---|---|---|---|---|---|---|---|---|
N | B | L | Y | Z | X | NifD | NifK | NifH | |
DPOR | |||||||||
N | 37–58 | 15–18 | 12–20 | ||||||
B | 34–62 | 15–22 | 14–18 | ||||||
L | 51–69 | 31–35 | 31–38 | ||||||
COR | |||||||||
Y | 35–78 | 13–15 | |||||||
Z | 39–81 | 11–16 | |||||||
X | 42–83 | 29–36 | |||||||
Nitrogenase | |||||||||
NifD | 17–70 | ||||||||
NifK | 37–58 | ||||||||
NifH | 67–75 |
6.
Rajesh Kasiviswanathan Matthew J. Longley Sherine S. L. Chan William C. Copeland 《The Journal of biological chemistry》2009,284(29):19501-19510
Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol γ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase γ (pol γ)3 bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol γ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3′ → 5′ exonuclease, and 5′-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol γ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol γ, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and recessive forms of progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, ataxia-neuropathy syndromes, and male infertility (tools.niehs.nih.gov/polg) (9).Alpers syndrome, a hepatocerebral mtDNA depletion disorder, and myocerebrohepatopathy are rare heritable autosomal recessive diseases primarily affecting young children (10–12). These diseases generally manifest during the first few weeks to years of life, and symptoms gradually develop in a stepwise manner eventually leading to death. Alpers syndrome is characterized by refractory seizures, psychomotor regression, and hepatic failure (11, 12). Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16, 17).During the initial cloning and sequencing of the human, Drosophila, and chicken pol γ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol γ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19–21). A high concentration of disease mutations, many associated with Alpers syndrome, is found in the thumb subdomain.Here we investigated six mitochondrial disease mutations clustered in the N-terminal portion of the polymerase domain of the enzyme (Fig. 1A). Four mutations (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) reside in the thumb subdomain and two (Q879H, c.2637g→t and T885S, c.2653a→t) are located in the palm subdomain. These mutations are associated with Alpers, PEO, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), ataxia-neuropathy syndrome, Leigh syndrome, and myocerebrohepatopathy (POLG mutation Disease Genetics Reference G848S Alpers syndrome In trans with A467T, Q497H, T251I-P587L, or W748S-E1143G in Alpers syndrome 15, 35, 43–50 Leigh syndrome In trans with R232H in Leigh syndrome 49 MELAS In trans with R627Q in MELAS 38 PEO with ataxia-neuropathy In trans with G746S and E1143G in PEO with ataxia 50 PEO In trans with T251I and P587L in PEO 51, 52 T851A Alpers syndrome In trans with R1047W 48, 53 In trans with H277C R852C Alpers syndrome In trans with A467T 14, 48, 50 In cis with G11D and in trans with W748S-E1143G or A467T Ataxia-neuropathy In trans with G11D-R627Q 15 R853Q Myocerebrohepatopathy In trans with T251I-P587L 15 Q879H Alpers syndrome with valproate-induced hepatic failure In cis with E1143G and in trans with A467T-T885S 35, 54 T885S Alpers syndrome with valproate-induced hepatic failure In cis with A467T and in trans with Q879H-E1143G 35, 54