1 Laboratory of Biocomputing, Centro Interdipartimentale per le Ricerche Biotecnologiche (CIRB), Bologna and Laboratory of Biophysics, Department of Biology, University of Bologna, Via Irnerio 42, 40126 Bologna, 2 IBPE, Consiglio Nazionale delle Ricerche, Via G. Marconi 10, 80125 Naples and 3 Università di Napoli `Federico II', Via Mezzocannone 16, 80134 Naples, Italy
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Abstract |
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Keywords: cross-linking/multiple sequence alignment/protein modeling/thermostable alcohol dehydrogenase/threading
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Introduction |
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The sequence of SsADH (347 residues) was determined by gene and peptide analysis (Ammendola et al., 1992). The tetrameric structure was defined by Cannio et al. (1996), who also reported the expression of the SsADH gene in Escherichia coli. Although SsADH is fairly resistant to denaturation at elevated temperatures, it shows a lower specific activity when compared with ADH from mesophilic or moderately thermophilic sources (Raia et al., 2001
). However, selective carboxymethylation of Cys38, a ligand of the catalytic zinc in SsADH, results in a more active although less stable enzyme (Raia et al., 1996
). On the other hand, the substitution of Asn249 with a tyrosine residue increases both SsADH activity and thermal stability (Giordano et al., 1999
). Preliminary crystallographic studies have been performed with apo and holo SsADH (Pearl et al., 1993
). However, the unsuccessful molecular replacement with crystals of apo-form and the twinning phenomenon which occurs on crystallization of the holo-form (Esposito et al., 1998
) hamper X-ray analysis.
This prompted us to develop a model of the tetrameric SsADH enzyme, which is described in this paper. Given the low sequence similarity of SsADH to the ADH structures currently known at atomic resolution, a procedure based on comparison (homology modeling) is not applicable. Our model is generated by adopting a threading procedure which includes constraints derived from both functional and structural characterization of the enzyme. In particular, we took advantage of the notion that SsADH contains two zinc atoms per subunit, with a catalytic and a structural function, respectively. In this respect SsADH is similar to the mesophilic dimeric two-zinc-containing horse liver enzyme (HLADH), although only 2425% identical in sequence (Ammendola et al., 1992). HLADH is known in two forms, apo and holo, depending on the absence or presence of bound NAD, which promotes a well documented rigid body inter-domain rotation of the catalytic domain with respect to the binding domain and a consequent closing of the inter-domain cleft (Colonna-Cesari et al., 1986
). However, although HLADH is the best homology model, it exists as a dimer. It was therefore necessary to use the structures of the two tetrameric ADHs in the PDB, from Thermoanaerobacter brockii (holo form only) (TbADH) and Clostridium beijerinckii (apo and holo forms) (CbADH), to model the quaternary organization, even though these enzymes share 2425% sequence identity with SsADH and contain only one zinc per subunit. Experimental results consistent with the model are described.
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Materials and methods |
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Dimethyl adipimate (DMA), dimethyl suberimidate (DMS) and ethylene glycol bis(succinimidylsuccinate) (EGS) were purchased from Pierce Chemical (Rockford, IL). Electrophoresis reagents and apparatus were obtainedfrom Bio-Rad (Hercules, CA). Other chemicals were A grade substances from Sigma Chemical (St. Louis, MO) or Applichem (Darmstadt, Germany).
Proteins
Preparation of coenzyme-free recombinant wild-type SsADH was performed as described previously (Giordano et al., 1999). For the cross-linking experiments, the SsADH apo form was dialyzed against 0.1 M TEAHCl, pH 8.5 and the holo form was prepared by adding to the latter 0.8 mM NAD.
Cross-linking
Cross-linking of SsADH with DMA, DMS and EGS was performed in 0.2 M TEAHCl, pH 8.5, for 1 h at 25°C with a final protein concentration of 0.2 mg/ml and a protein to bifunctional reagent ratio of 1:6.2 and 4.4 (µg/µg). Reactions were stopped by denaturing the samples for 15 min at 90100°C in the presence of 12% SDS and 2-mercaptoethanol. Gel electrophoretic separation was performed essentially as described (Davies and Stark, 1970) using 5% polyacrylamide rod gels. Amounts of 210 µg of protein were loaded on to each disk gel. Protein bands were visualized by staining with Coomassie Brilliant Blue G-250 and quantification of the band intensity was achieved using a Bio-Rad Model GS-710 imaging densitometer.
The spacer lengths of the bifunctional reagents DMA, DMS and EGS are 8.6, 11.0 and 16.1 Å, respectively. Owing to the flexibility of the polypeptide chain and the limited accuracy of the model, it is assumed that each reagent can span two neighboring lysine residues provided that their separation in the model is within ±2 Å, the length of the spacer between two reacting groups. Moreover, it is assumed that all Lys residues involved in cross-linking have the same reactivity.
Multiple sequence alignment
All the sequences of two zinc-containing ADHs were searched in the SwissProt database (Release 37, December 1998). The resulting 87 sequences were aligned with CLUSTALW (Thompson et al., 1994). The residues involved in the binding of the two zinc atoms were deduced from this alignment. Similarly, the sequences of 14 tetrameric ADHs were searched and aligned to extract the sequence features that contribute to the oligomerization.
Structural alignments
The 3D crystal structures of ADHs were extracted from the PDB files and were superimposed with the BRAGI program (ftp.gbf.de/pub/Bragi) to obtain pairwise structural alignments.
Secondary structure prediction
The secondary structure of SsADH was predicted with a program based on neural networks (Jacoboni et al., 2000) and with a consensus based procedure (Cuff and Barton, 1999
); the secondary structural elements of the known ADHs structures were defined using the DSSP program (Kabsch and Sander, 1983
). When necessary, solvent exposure of residues was evaluated with the DSSP program.
Model building and evaluation
The comparative model building was performed with the MODELLER program (ali and Blundell, 1993
). For a given alignment, 10 model structures were built and were evaluated with the PROCHECK suite of programs (Laskowski et al., 1993
). Only the best evaluated model was retained after the analysis. Tetrameric assemblages were obtained by superimposing with the BRAGI program the modeled monomer with each subunit of the tetrameric templates. Close contacts were removed with the DEBUMP program of the WHATIF package (Vriend, 1990
).
Docking of the coenzyme
Docking simulation of NADH coenzyme into the monomer was performed with the AutoDock 3 program, which allows flexible docking by means of Lamarckian Genetic algorithms (Morris et al., 1998). Fifty independent runs were performed, each one processing a population of 100 conformations for 27000 generations with rates of mutation and crossover set to 0.02 and 0.8, respectively. The elitism parameter was set to 2. The docked conformation with the lowest value of estimated free binding energy was retained for further analysis. In order to compute the parameters to be used in docking the coenzyme to the SsADH holo model, the binding of NADH in the site of HLADH was performed. The result agrees with the conformation of co-crystallized coenzyme (PDB file 2OHX): the root mean square deviation (r.m.s.d.) value between the real and the docked conformations is 0.95 Å; the energy of interaction is -20.76 kcal/mol (-21.93 kcal/mol from intermolecular interactions, 1.18 kcal/mol from intramolecular interactions) and the free energy of binding is -16.64 kcal/mol, as estimated by the program AutoDock 3.
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Results and discussion |
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A key feature of our modeling procedure stems from the following considerations: SsADH is a two-zinc-containing protein with low sequence identity (25%) to all the other ADH sequences of known 3D structure; the functional unit of the target is a homotetramer; and the enzyme can bind NADH. In this respect, one should consider that the PDB database contains 31 atomic resolved structures of ADHs, corresponding to only eight different sequences from different species; 28 of these structures (six sequences) are two-zinc-containing dimeric ADHs, while the remaining three are one-zinc-containing tetrameric ADHs. There is no two-zinc tetrameric ADH structure available. Furthermore, 26 of these structures are holo and two-zinc dimers; two are apo and two-zinc dimers; two are holo and one-zinc tetramers; and one is apo and one-zinc tetramer. An interesting observation useful to our procedure is that the functional set of ADHs has monomers (containing either two or one zinc ions) whose structure is within 1.8 Å of the r.m.s.d. (data not shown). This confirms that ADH monomers have a rather well conserved structure irrespective of the level of sequence identity of the different species, number of zinc ions and aggregation state of the monomeric unit. This observation makes it highly probable that the monomer of our target will adopt a similar backbone conformation. Furthermore, if we require that the template structure has two zinc ions and is endowed with both the holo and apo forms, we are forced to adopt only HLADH as a template. However, the aggregation state of HLADH is dimeric whereas we know from experimental observations that our target is tetrameric and presumably with inter-monomer interactions different from those of the dimer. Therefore, for monomer aggregation we should adopt different targets, with a tetrameric aggregation state. A solution to this problem can be to search the ADH database of sequence and structures to find residues in the target which are conserved both in the two-zinc-containing and in the tetrameric enzymes. This would constrain threading of the target to the templates.
Multiple sequence alignment
In order to cope with our requirements, from the 90 ADH sequences contained in the SwissProt database, 87 were selected as containing two zinc ions. Multiple sequence alignment highlights that 38 residues are conserved in more than 90% of the 87 sequences (in Figure 1, only 20 out of the 87 aligned sequences are shown for practical reasons). Cys38, Gly67, His68, Glu69, Gly72, Gly87, Cys101, Cys104, Cys112, Cys154, Gly184 and Gly216 (numbering according to SsADH) are conserved in all the sequences. Cys38, His67 and Cys154 constitute the binding site of the catalytic zinc. Glu98, Cys101, Cys104 and Cys112 are the residues involved in the binding of the structural zinc atom. Glu98 is substituted in all the other sequences with Cys (Vallee and Auld, 1990
). Theoretical and experimental approaches have shown that this mutation does not affect the binding of a zinc atom and contributes to the thermostability of the enzyme, possibly by influencing the electrostatic interactions of the binding site (Ammendola et al., 1995
). Overall, 28 of the 38 highly conserved residues are found in the SsADH sequence; 11 of them are glycines that can be markers of the presence of a loop in the structure. This first step constrains the residues involved in binding both the structural and catalytic zinc ions.
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The results described above prompted us to adopt two sets of template structures, since we are interested in modeling both the holo and apo forms of SsADH. We considered as prototypes for the holo form the 1.8 Å resolved structure of the dimeric holo-HLADH (PDB code: 2OHX) and the structure of the tetrameric TbADH solved in the holo form at 2.5 Å (PDB code: 1YKF). The structure of the apo HLADH (PDB code: 8ADH, 2.4 Å resolution) and the structure of the apo tetrameric CbADH (PDB code: 1PED, 2.15 Å resolution) were used as templates for the apo form. The sequence of CbADH is, however, 75% homologous with that of TbADH (Korkhin et al., 1999). Structural alignment between the two sets of templates indicates that the main difference between HLADH and the two bacterial enzyme structures is the conformation of the loop coordinating the structural zinc ion, present only in HLADH. Moreover, loop 114143 in HLADH differs considerably from the corresponding loop 105117 in TbADH and CbADH. The first one is longer and comprises two strands, whereas the others are unstructured (Figure 3
).
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In order to align the target structure with the templates, we first predicted the secondary structure of the target with well consolidated methods of protein secondary structure prediction [neural network-based and implemented in house (Jacoboni et al., 2000); the results were also compared with a method based on a consensus procedure (Cuff and Barton, 1999
)]. The target sequence is then aligned with the structural alignment of the templates taking into consideration the information derived from the multiple sequence alignments described above (Figure 3
) and from the secondary structure prediction. This procedure is essentially an expertise-driven one, satisfying the constraints of the two-zinc binding sites and those of the tetrameric form. The loop coordinating the structural zinc atom in SsADH (residues 98110) is modeled only on the structure of HLADH. The loop, which is similar in the two tetrameric bacterial structures and differs in HLADH, is aligned to exclude the corresponding portion of the HLADH chain. This is based on the finding that multiple sequence alignment and secondary structure prediction indicate that the corresponding segment on SsADH (residues 115123) is more similar to the tetrameric structures.
The segment comprising residues 5260 of SsADH is not aligned with any template sequence. This is an insertion in an exposed loop of the templates and its structure is automatically built and optimized by the modeling algorithm.
The procedure outlined above is similar for modeling both the apo and holo forms of SsADH.
The monomer model
The low-resolution model that we obtain after energy minimization is the monomer unit, which comprises two domains, separated by a deep cleft where the catalytic zinc atom is bound. The quality of the model was checked with Procheck (Laskowski et al., 1993).
Characteristic features derived from the templates are as follows: the coenzyme binding domain (residues 156295) is a Rossmann fold; the catalytic domain (residues 1155 and 296347) is an all-ß GroES-like fold; and 27% of the residues are in helical structure and 20% are in a ß-strand conformation. The structural zinc atom is bound in an external loop (Figure 4).
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A salient feature of the model is that the two tryptophan residues contained in the monomer are buried (Trp95 and Trp117), in both the apo and holo forms, as detected by measuring the relative solvent accessibility. This is consistent with SsADH intrinsic fluorescence studies, showing that the fluorescence spectrum of enzyme is a relatively blue one with max = 321 and 319 nm for the apo and holo form, respectively (Giordano et al., 1999
).
The tetramer model
The tetrameric organization is built automatically by BRAGI, by performing structural alignment of the apo and holo forms with the monomer of the tetramers selected as targets in our modeling procedure: CbADH for the apo form and TbADH for the holo form, respectively. The tetrameric organization consists of two interacting dimers in which interactions between ß-strands in positions 289294 and between stretches 273 and 282 stabilize monomermonomer interaction (Figure 5).
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Interestingly, the loop containing the structural zinc ion is less solvent-exposed in the holo than the apo form. The solvent accessibility values (in Å2) of the side chain of the four structural zinc ligands are the following: Glu98 70 and 24, Cys101 10 and 9, Cys104 0 and 0 and Cys112 0 and 0 for the apo and holo form, respectively. Glu98 was replaced by a cysteine residue by site-directed mutagenesis (Ammendola et al., 1995), thus restoring the structural zinc binding site of mesophilic ADHs, which is characterized by four cysteine residues (Vallee and Auld, 1990
). The Glu98CysSsADH mutant proved equally active but less thermostable than native SsADH, suggesting that at least part of the SsADH thermostability is due to the presence of the glutamate in its structural metal binding site. The higher accessibility of Glu98 residue with respect to the other three ligands supports the hypothesis that the replacement of Cys by Glu represents a significant achievement in the evolutionary adaptation of alcohol dehydrogenase to thermophilic conditions (Ammendola et al., 1992
).
Glu108, Glu109, Lys263 and Lys266 of the monomers of each dimer are ion-bridged in both the apo and holo forms. The two dimers interact with four different surface patches: the first includes residues 301309, 170173 and 193198 of subunit A and interacts with the same residues of subunit D; the second includes the same segments between the B and C subunits; the third patch is composed of residues 96110, 135137 and 301299 of subunit A which interacts with the corresponding surface of subunit C; the fourth includes the same segments between the B and D subunits.
Twenty more ion-bridges stabilize the tetramer of the holo form: Glu98 and Lys136 of subunits A and C interact and similarly those of subunits B and D; the couples Asp218Lys313, Asp218Arg307, Asp242Arg307 and Asp169Arg164 can be detected between the subunits A and D and the subunits B and C.
In the apo form, 16 more interdimeric ion-bridges are detectable: Glu98Lys136 which stabilize A with C and B with D; Asp169Arg164, Asp169Arg161 and Asp218Arg307 are detected between subunits A and D and subunits B and C.
The overall electrostatic and chemico-physical properties of the model are listed in Table I. The elevated number of ion pairs suggests that SsADH retains its enzymatic activity at higher temperatures because of greater stability of subunit interaction in the tetramer. Moreover, the additional ion pairs in the holo enzyme strongly agree with the experimental observation that holo- is less sensitive than apo-SsADH to the disaggregating action of protein denaturants (Giordano et al., 1999
; Raia et al., 2001
).
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Binding of NADH
We estimated NADH binding to the holo form with a docking procedure. The result indicates that NADH binds to its pocket with an interaction rather similar to that detected in the holo form of HLADH (2OHX). The r.m.s.d. value to the ligand in HLADH is 1.4 Å (Figure 6). The interaction energy of the docked conformation of NADH is -22.30 kcal/mol (-23.74 kcal/mol intermolecular, 1.45 kcal/mol intramolecular) and the estimated free energy of binding is -18.44 kcal/mol. Comparison of this value with that estimated for HLADH, -16.64 kcal/mol, suggests a tighter binding of the coenzyme to the archaeal than mammalian ADH. Interestingly, the Kd values are 0.02 and 1.1 µM for the SsADHNADH and HLADNADH complex, respectively (Iweibo and Weiner, 1972
; Raia et al., 2001
). Asp203 binds the adenine ribose and so it determines the specificity for NADH with respect to NADPH (similarly to Asp223 in HLADH and yeast ADH; Fan et al., 1991
). Nicotinamide ribose interacts with Asn248 and Asn249 (Figure 6
). It was previously suggested that Asn249Tyr substitution increases SsADH activity, possibly by decreasing ligand-binding affinity (Giordano et al., 1999
). This finding in the model supports the early hypothesis that Tyr249 sterically and electrostatically contrasts the interaction of the coenzyme with the binding site. Trp95 is found at a 6 Å distance from the nicotinamide moiety of the reduced coenzyme. This is consistent with the very intense energy transfer band centered at 422 nm occurring upon addition of only a stoichiometric amount of NADH (Giordano et al., 1999
) and the hypothesized location of the Trp95 residue at the catalytic site (Raia et al., 1996
).
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The SsADH molecule contains 23 lysine residues per subunit, most of which change significantly their solvent exposure and reciprocal intra- and intersubunit distances upon the rearrangement of the apo to the holo form, as indicated by the different solvent accessibility values (data not shown). This feature can be tested using a cross-link methodology with the aim of evaluating the differences in through-space distance between lysine residues and indirectly to assess the consistency of the tetramer assembly.
A measure of the lysine pair distance in the model indicates that below a cut-off distance of 14 Å only AD and BC dimers can be cross-linked, provided that a cross-linker of a suitable length is used. At greater distances (>14 Å), cross-linking of lysine pairs in the AB and DC dimers makes the formation of tetramers more likely.
The apo and holo forms have six (K136, -140, -192, -213, -239 and -313) and three Lys residues (K192, -213 and -313), respectively, which are solvent exposed and suitable to be conjugated by bifunctional reagents spanning up to 14 Å. Only AD and BC dimers and no tetramer can be formed through these residues pairs. This suggests that apo SsADH will yield more cross-links than the holo enzyme and that the production of tetramer will be due only to intermolecular cross-linking.
According to our model, four Lys residues of the apo (K266, -172, -24 and -136) and only two Lys residues of the holo form (K136 and -24) are found at a distance of about 16 Å and solvent exposed. Their cross-linking can probably give tetramers.
Samples of SsADH were treated with bifunctional reagents spanning a distance from about 8 to 16 Å in the presence and absence of NAD and the resulting reaction mixtures were then studied by SDS electrophoresis (Figure 7). Quantification of the band intensity (Table II
) shows that the extent of cross-linking achieved with the three reagents is significantly higher for the apo than the holo form of SsADH and that the cross-linked tetramer is the predominant species only with the longer reagent EGS, in agreement with the theoretical prediction gathered from our SsADH model.
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To our knoweledge, this is the first report of a modeling procedure of a tetramer of a chain with low sequence identity with the structures of the database and the first model of a two-zinc-containing tetrameric ADH. Our threading procedure takes into consideration the following key observations: (1) all the monomers of the ADHs present in the database can be structurally aligned with an r.m.s.d. of 1.8 Å, indicating that in spite of different sources and number of bound metal ions (one or two zinc ions), the monomeric unit is structurally well conserved independently of the aggregation state (dimers or tetramers); (2) residues coordinating both the catalytic and structural zinc ions are well conserved in the multiple sequence alignment; (3) residues at the monomer interface in sequences annotated as tetramers in the sequence database are conserved in the multiple sequence alignment.
The computed model is a low-resolution model whose C backbone is within 2 Å from the ADH monomers of the structure database. However, docking of NADH to the binding site reveals that this portion of the protein is computed with an accuracy sufficient to dock the ligand as in HLADH. Static fluorescence data and energy transfer measurements, previously performed on the same protein, are consistent with the model. Cross-linking experiments are in agreement with predictions of dimer and tetramer formation as evaluated by measuring the lysine pair distances in the model.
Although the experimental data only support and do not fully validate the SsADH model, our computational approach is strengthened by the critical assessment that residues are evolutionarily conserved at both the catalytic and structural zinc ion binding sites and at the monomer interface.
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Notes |
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Acknowledgments |
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References |
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Received April 17, 2001; revised November 21, 2001; accepted December 7, 2001.