1 Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF, Bidhan Nagar, Calcutta 700 064 and 2 Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India
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Abstract |
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Keywords: reactive-site loop/site-directed mutagenesis/winged bean chymotrypsin inhibitor/X-ray structure
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Introduction |
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In our previous structural studies on WCI, it was observed that the side chain of one N-terminal residue, Asn14, intrudes inside the first reactive-site loop (Figure 1A) and forms a network of hydrogen bonds involving the main-chain and the side-chain atoms of loop residues (Dattagupta et al., 1996
, 1999
).
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In this study, we wanted to see the effect of the conserved Asn residue on the stability and functionality of the first reactive-site loop of WCI. First, the cDNA of WCI was cloned (Ghosh and Singh, 1997) and Asn14 was mutated to Lys (N14K) and Asp (N14D) individually through site-directed mutagenesis. These mutations (Asn14
Lys and Asn14
Asp) were done with a view to observing the spatial and electrostatic effect of side chains at this conserved position. Although these are point mutations, by virtue of the crucial position of Asn14 and its hydrogen-bonding interactions with neighboring loop residues, they are nonetheless useful for giving an idea about the contribution of the scaffold in the stability of the reactive-site loop. The crystal structure of recombinant WCI and its two mutants N14K and N14D were determined and their structural characteristics compared. The process of mutation, expression, purification and the results of biochemical assay are also reported.
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Materials and methods |
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The double-stranded cDNA pool synthesized by reverse transcriptase and DNA polymerase I from the poly(A)+ RNA of developing winged bean seeds has been used for PCR amplification of the WCI gene (Ghosh and Singh, 1997). The recombinant pBS-WCI was subjected to PCR-directed mutagenesis using mutagenic oligonucleotide primers containing single base mismatches (Higuchi, 1990
; Zhao et al., 1993
). The mutagenic PCR involved the generation of two PCR products that overlap in sequence containing the same mutation introduced as part of the PCR primers. A subsequent re-amplification of these fragments with cloning primers resulted in the enrichment of the full-length secondary product. A single (T/A) transition in the AAT codon generated by this method will code for Lys instead of Asn14 when expressed upon induction by IPTG. Similarly, substitution of the first A for G in the same codon will have Asp in the same position in the expressed protein. The plasmids harboring the mutated genes were named pBS-N14K-WCI and pBS-N14D-WCI, respectively.
For the generation of DNA ends encoding the N-terminal part of the protein from the site of mutation, the M13 reverse primer and the primer with sequences corresponding to the opposite strand of the DNA containing the desired point mutation (N14K BKW and N14D BKW) were used. Again, for the amplification of C-terminal part of the protein, the mutagenic primer with the single base substitution (N14K FWD and N14D FWD) and the M13(40) primers were used. Taking these two PCR products as template, another set of PCR was performed using WCI FWD and WCI REV primers to generate EcoRI and SalI sites and the PCR product thus obtained was cloned into pBS-SK(). All these products were sequenced using an ABI 377 automated DNA sequencer.
Sequences of primers used for mutation of Asn into Lys and Asp
The sequences were as follows:
N14K FWD 5'-GGTAACTTAGTTGAAAAAGGTGGCACA-3'
N14K BKW 5'-TGTGCCACCTTTTTCAACTAAGTTACC-3'
N14D FWD 5'-GGTAACTTAGTTGAAGATGGTGGCACA-3'
N14D BKW 5'-TGTGCCACCATCTTCAACTAAGTTACC-3'
WCI FWD 5'-GG-GAATTC-GATGATGATTTGGTCGATGC-3'
WCI REV 5'-GAGAGA-CTCGAG-GGATGAGAAGTGCTTAATGGC-3'
The sequence within the dashed region in the WCI FWD primer denotes the EcoRI recognition sequence and that in the WCI REV primer shows the SalI recognition sequence. In the top four primers, the single bases, denoted by bold letters, represent the point mutations over the WCI gene sequence.
For bacterial overexpression of N14K and N14D, these sequences were subcloned into pTrc99A (Amersham Pharmacia Biotech) and induced with 0.1 mM IPTG for 1 h at 37°C. A 2.5 g amount of optimally induced E.coli XL1-Blue cells, harboring the chimeric plasmids, was sonicated and the soluble fraction was used for the purification of mutant proteins by single-step immunoaffinity chromatography, with an overall yield of ~98% (see Table I). The same procedure was used for the recombinant protein (rWCI) and the overall yield was similar (Ghosh and Singh, 1997
). In all three cases, the specific protein content was nearly one and usually the preparations were essentially homogeneous as attested by single protein bands in SDS-PAGE. However, unlike rWCI and N14K (Figure 2
, lanes b and d), in the case of N14D mutant, there were some faster moving minor bands in addition to major band (Figure 2
, lane c). These were identified by immunoblot analysis as fragments of the purified protein. The proteins were quantitated using the Bradford method (Bradford, 1976
).
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Crystals of rWCI and the two mutants were grown according to the procedure mentioned in the crystallization of native WCI (Dattagupta et al., 1999). However, the reservoir solution contained 30 and 35% (w/v) ammonium sulfate (0.1 M acetate, pH 5.4) for rWCI and mutants, respectively. X-ray diffraction data were collected at the ID14-EH4 undulator MAD beamline at ESRF (Grenoble, France). First, the crystals were exposed briefly (100 ms) in order to derive the crystal quality and its orientation. Then the oscillation range was maintained at 0.5° (
t = 1 s/frame and for low resolution
t = 0.1 s/frame) and the final datasets were collected. They were processed and scaled using MOSFLM (Leslie, 1990
) and SCALA (Evans, 1997
), respectively. The scaled intensities were converted to structure factor amplitudes using TRUNCATE (French and Wilson, 1978
). The crystals belong to the hexagonal space group P6122. Data collection statistics are given in Table II
.
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The coordinates of native WCI (PDB entry 4WBC; Ravichandran et al., 1999) served as the starting model for the refinement of rWCI and the two mutants. However, the disordered regions (His137Asp142, Lys176His183) and the solvent molecules were excluded from the starting model. Also, in order to avoid model bias in the N-terminal region (since the cloned proteins have the additional tripeptide sequence Met1'Glu2'Phe3'), residues Asp1 and Asp2 were excluded from the starting model. For the mutants, Asn14 was replaced by Ala in the starting model. All initial calculations were done using X-PLOR (Brünger, 1992) and CNS (Brünger et al., 1998
). For model building, the programs TOM (Jones, 1985
) and O (Jones et al., 1991
) were used.
Refinement of rWCI
In the case of rWCI, 15 cycles of rigid-body refinement followed by a simulated annealing procedure (Brünger et al., 1990) of slow cooling from a starting temperature of 3000 K to 300 K in steps of 25 K yielded a starting R factor of 36.7% (Rfree = 40.1%). The next 75 cycles of positional refinement involving Powell minimization further reduced the R factor to 33.4% (Rfree = 36.8%).
A weighted maps (Read, 1986
) with coefficients 3m|Fo| 2D|Fc| and m|Fo| D|Fc| were then calculated. Water molecules were gradually incorporated and a flat bulk-solvent correction (Jiang and Brünger, 1994
) was applied in all subsequent stages of the refinement. These water molecules were added to the coordinate list only if a peak greater than 3
appeared in the Fo Fc difference map and the position of the peak implied at least one hydrogen bond within 3.6 Å, either to a protein atom or to an already defined water molecule. At this stage, a few strong peaks (>4
height) were observed and were attributed to sulfate ions based on the chemical environment and tetrahedral shape of the electron density. Following several cycles of positional and individual temperature factor refinement, the R factor dropped to 23.7% (Rfree = 27.1%). At this stage, the refinement was continued using the programs REFMAC (Murshudov et al., 1997
) and wARP (Lamzin and Wilson, 1993
). More water molecules were added in the model and, finally, all solvent positions were re-confirmed using an OMIT map (Bhat, 1988
) involving a modified
A coefficient (Vellieux and Dijkstra, 1997
). Residues Met1' and Glu178His183 were not included in the final model owing to ill-defined electron density around these regions. For the same reason, the N-terminal residue (Glu2') was kept as Ala in the final model. At the end of final refinement, the R factor converged to 19.5% (Rfree = 24.6%) for 16 825 reflections in the resolution range 10.01.9 Å.
Refinement of N14K and N14D
In case of the mutants, 40 steps of rigid body refinement, followed by 75 steps of positional refinement yielded a starting R factor of 38.1% (Rfree = 41.9%) and 28.2% (Rfree = 31.2%) for N14K and N14D, respectively. Lys and Asp were incorporated in the corresponding models to satisfy the extra electron density at residue 14, which was Ala in the starting model. All solvent molecules were identified and located using the criteria mentioned before. Next, 100 steps of positional refinement and 120 steps of simulated annealing (slow cool) refinement further reduced the R factor (N14K, R = 30.6%, Rfree = 39.0%; N14D, R = 26%, Rfree = 30.0%). A maps were then calculated and the models were re-built. After a few cycles of positional and individual B factor refinement, the R factor further dropped to 22.6% (Rfree = 25.5%) and 24.1% (Rfree = 29.1%) for N14K and N14D, respectively. In order to confirm the correct side-chain orientation of the mutated (fourteenth) residue and to resolve any ambiguity or model bias, OMIT maps were calculated for the two models. During this procedure, the mutated residue was omitted and a couple of other regions (Lys176Ser177 and His137Asp142), where high atomic B factors were noted, were also omitted in the two models. Finally, a few cycles of maximum likelihood (MLKF) refinement were performed using REFMAC. The refinement statistics for the final models of N14K and N14D are shown in Table III
. Owing to the poor quality of the electron density map, residues Met1' and Glu178His183 could not be located in the final model of N14K as per the wild type. Also for the same reason, residues Met1'Glu2'Phe3' and Glu178His183 could not be located in the final model of N14D.
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Results |
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The purified proteins, recombinant and mutants, were subjected to biochemical assay. rWCI, purified from bacterial lysate, was found to be as active against -chymotrypsin as the naturally occurring WCI, the respective specific activities being 69 and 74 units/mg protein with N-benzoyl-L-tyrosine p-nitroanilide used as substrate; it also inhibits the proteinase in the same non-stoichiometric pattern as WCI. The specific inhibitory properties of the mutant inhibitors were then studied with
-chymotrypsin and were found to follow the same non-stoichiometric inhibition pattern as observed in the case of rWCI (Figure 3
). The observed pattern of inhibition was clearly suggestive of differential strength and stoichiometry of binding of different inhibitors. Whereas the rWCI molecule was observed to inhibit
-chymotrypsin in a 1:2 ratio as seen in the figure, the N14D molecule showed an almost 1:1 inhibition ratio and N14K molecule showed an intermediate pattern. The specific activity of the mutant inhibitors as well as their dissociation constants with the enzymes were also determined as described by Bieth (1974). The specific activities were 4800 units/mg protein for rWCI, 2900 units/mg protein for N14K and 1500 units/mg protein for N14D, using BTEE as substrate. Again, for the non-stoichiometric inhibition pattern of these inhibitors, the dissociation constants (Ki) of these inhibitors were calculated using the equation
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where [E], [I] and [EI] are the molar equilibrium concentrations of enzyme, inhibitor and the complex, respectively. Now, if the stoichiometry of association is different from 1:1, then assuming the binding sites to be equivalent and independent, the intrinsic dissociation constant can be determined by the equation
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where n is the number of binding sites for the enzyme, which was taken as two in this case, [E0] and [I0] are the total concentrations of the enzyme and inhibitor, respectively, and a represents the fraction of the enzyme not bound to the inhibitor. The Ki values, obtained from the solution of the equation, for rWCI, N14K and N14D were 1.14x109, 2.4x109and 2.58x109 M, respectively. The stoichiometry of chymotrypsininhibitor complex formation was studied for WCI, rWCI, N14D and N14K by gel permeation chromatography in an FPLC system with a Superose 12 HR 10/30 column. All four inhibitors formed both 1:1 and 1:2 complexes and, in this respect, they were indistinguishable.
Structural comparison of rWCI with native WCI
The three-dimensional structure of rWCI was first compared with the 2.13 Å model of native WCI and, as expected, there was no significant deviation in the overall topology of the two models. The superposition calculations were performed using the programs LSQKAB (Collaborative Computational Project Number 4, 1994) and TOP (Lu, 2000
). The main-chain backbone atoms of the 177 amino acid residues superpose with an r.m.s. deviation of 0.5 Å while all the atoms, including the side-chain atoms, superpose with an r.m.s. deviation of 0.86 Å. However, minor conformational changes were observed in a few loop regions and the most notable change was at the ß-turn region, Pro70Ser73, where a `peptide flip' occurred. This region of rWCI adopts a type I ß-turn conformation (
71 = 67.9°;
71 = 17.0°;
72 = 89.2°;
72 = 9.7°), whereas in the native WCI model, the corresponding region had a type II conformation (
71 = 54.1°;
71 = 130.5°;
72 = 82.8°;
72 = 7.6°). Such an interconversion phenomenon, in general, depicts a stereochemically mobile site in homologous crystal structures (Gunasekharan et al., 1998
). In addition, conformational changes were also observed in the N-terminal region due to the presence of the extra tripeptide sequence (Met1'Glu2'Phe3') in rWCI. The average r.m.s. deviation for the two N-terminal residues (Asp1 and Asp2) was 2.2 Å and the carbonyl group of Asp1 showed an ~180° peptide flip in this region. Also, double occupancies for the side-chain OG atom of two serine residues, Ser66 and Ser73, were observed (Figure 4
). These were not seen in the native WCI model.
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An overall structural comparison of the mutant proteins with rWCI reveals that the mutation at this conserved position had almost no effect on the overall topology of the protein. The main-chain CA atoms of 176 amino acid residues of N14K and N14D superpose on rWCI with r.m.s. deviation values of 0.2 and 0.4 Å, respectively. Figure 5 shows the electron-density map around the side chain of the fourteenth residue in the final models of rWCI, N14K and N14D. A superposition of the first reactive-site loop of N14K and N14D on that of rWCI is shown in Figure 6
. The r.m.s. deviation values, obtained from CA superpositions of loop residues, are 0.09 and 0.27 Å for N14K and N14D, respectively. In all three structures, the reactive-site loop maintains more or less an identical canonical conformation (Bode and Huber, 1992
). The values of dihedral angles (
,
) for the loop residues have been compared in Table IV
.
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Discussion |
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For the Kunitz (STI) family of serine protease inhibitors, the role of a conserved Asn residue coming from the N-terminal segment was emphasized owing to its crucial position (Onesti et al., 1991; Meester et al., 1998
). In the present study on rWCI, it is observed that the interactions made by the side chain atoms of Asn14 are more pronounced than its main-chain atoms in stabilizing the conformation of the reactive-site loop. Figure 7A
shows the hydrogen-bonding pattern around the first reactive-site loop of rWCI. The side-chain amide group of Asn14 makes three major hydrogen bonds with the reactive-site loop residues: Asn14 ND1 with carbonyl O atoms of Phe64 (P2; 2.91 Å) and Ser66 (P1'; 2.86 Å) and Asn14 OD2 with the main-chain N atom of Ser62 (P4; 2.80 Å); whereas the main-chain N atom of Asn14 contributes only one, i.e. with the carbonyl O atom of Leu67 (P2'; 2.81 Å). This was also observed in the crystal structure of native WCI (Ravichandran et al., 1999
). Amongst these, the hydrogen bond between the Asn14 side chain and the P2 residue (Phe64) is conserved in this family. For example, in ETI, Asn12 ND1 makes a hydrogen bond with the carbonyl O of Leu62 and in STI, Asn13 ND1 makes a hydrogen bond with the carbonyl O of Tyr62. A similar interaction is also observed in PSTI, a member of Kazal family, where Asn33 ND1 makes a hydrogen bond with the carbonyl O of P2 residue. Thus, it has been proposed that this conserved asparagine acts as a `fixed spacer' to keep the loop in a protruded conformation (Papamokos et al., 1982
; Onesti et al., 1991
).
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To check whether Asn14 in WCI is similarly responsible for stabilizing the binding loop conformation, it was mutated to Lys and Asp. Such mutations may help in understanding the characteristics of this crucial residue, contributed by the scaffolding of the inhibitor. A long side chain such as Lys, in place of Asn14, was expected to disturb the stabilizing interactions with the loop. Similarly, a negatively charged residue such as Asp in such a position was also expected to disrupt the hydrogen-bonding pattern.
The first reactive-site loops of N14K, N14D were analyzed and compared with that of rWCI. Figure 7B and C show the hydrogen-bonding pattern around the first reactive-site loop of N14K and N14D, respectively. In rWCI (Figure 7A
), apart from the Asn14 side-chain interactions mentioned above, additional stability comes from two water molecules (Wat260 and Wat241) located near the reactive-site loop. Wat260 forms hydrogen bonds with the carbonyl O atoms of P4 (Ser62) and P2 (Phe64) residues while Wat241 is hydrogen bonded to the side-chain OG atom of Ser62 and the carbonyl O atom of the P3' residue (Phe68). In the mutants the conserved hydrogen bond between the fourteenth residue side chain and P2 residue is absent. However, as in rWCI, the main-chain N atom of the fourteenth residue of both mutants maintains a similar pattern of hydrogen bonds with the P2' residue (Leu67). Besides this, there is a water molecule, Wat350 in N14K, which corresponds to Wat260 of rWCI and shows a similar pattern of hydrogen bonding, whereas no water molecule corresponding to Wat241 of rWCI is observed. The orientation of the long side chain of Lys14 is responsible for the absence of this water molecule. When superposed with rWCI, the side-chain NZ atom of Lys14 is found to occupy the corresponding position of Wat241 and makes hydrogen bonds (of distance 2.33 and 2.63 Å, respectively) with side-chain OG atoms of two serine residues (P4 and P1') and the carbonyl O-atom of Phe68 (P3'; 2.61 Å). In N14D, the hydrogen-bonding network at the loop region is mediated through a water molecule, Wat248. The position of this water molecule corresponds to the side-chain ND1 atom of Asn14 in rWCI (see Figure 7A
). Wat248 forms hydrogen bonds with the main-chain N and carbonyl O atoms of Ser66 (P1'; 3.18 and 2.96 Å, respectively), the carbonyl O atom of Phe64 (P2; 2.74 Å) and the carboxylate oxygen atoms of Asp14 (2.46 Å) (Figure 7C
). Such water-mediated interaction between the reactive-site loop and the protein scaffold was also observed in a few protease inhibitors, e.g. in BPTI, where the interaction is mediated through a water molecule to Gly12 (Otlewski et al., 1999
). Apart from this, one of the conserved water molecules (Wat260 of rWCI) is absent in N14D. This is because of one of the alternating positions of the side-chain OG atom of Ser61 (P5) has occupied this region.
Finally, our observations point to the fact that the hydrogen-bonding interactions are almost conserved in rWCI, N14K and N14D. In N14K, although the conserved hydrogen bond involving the side chain of the fourteenth residue and the P2 carbonyl oxygen is not observed, the canonical conformation of the loop remains intact. The long side chain of Lys14, instead of destabilizing the conformation of the reactive-site loop, has itself folded back with an unusual non-rotameric fold. It is seen that the accessible surface area for the Lys14 side chain is 2.44 Å2 and this value is the lowest among the values calculated for the remaining Lys side chains in the model. Moreover, it is reported that Lys side chains can display environment-dependent conformational changes and their flexibility becomes higher during ligand binding to proteins (Najmanovich et al., 2000). Hence it can be understood that the inherent conformational flexibility of the Lys side chain and its limited accessible surface area are responsible for the unusual fold of the Lys14 side chain.
From consideration of the retention volumes of the complexes, it is evident that all three inhibitors form both types of complexes (1:1 and 1:2) with chymotrypsin, depending on the stoichiometry of the incubation mixture. This means that the replacement of Asn14 with Lys or Asp has only a minimal effect on the binding property of the inhibitor molecules. However, minor changes in the hydrogen-bonding pattern could be a plausible reason behind the 2-fold alterations in the dissociation constant values. This result also agrees with the observation of a scissile bond hydrolysis experiment performed for a point mutant, N33S of ovomucoid third domain (a Kazal family inhibitor), where the change in hydrolysis constant Khyd was found to be relatively small, i.e. not exceeding a factor of 35 (Ardelt and Laskowski, 1991). The conformational stability of the reactive-site loop and its electrostatic environment therefore depends mainly on its residue type and the local interactions. The residues of the loop (Gln63Phe68) demand a hydrogen-bonding network for its stability and this need is satisfied in all the structures either by the residues of the scaffold or by solvent molecules keeping the loop conformation intact. As a result, the activity of the protein remains almost unaltered. In other words, the replacement of Asn14 with polar residues marginally influences the activity but its actual role, however, can only be estimated after drastic mutations at this site.
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Notes |
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Acknowledgments |
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References |
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Received September 6, 2000; revised January 22, 2001; accepted February 15, 2001.