2.4 Results and discussion
3.4.2 KlenTaq M747K in ternary complex with p/t, ddCTP and dCMeTP
Soaking experiments with dCMeTP and dCEtTP were done, to see how the sterically 4’-methyl- or 4’- ethyl- modified deoxycytosine triphosphates are incorporated and how they fit in the active center. To remove all bound ddCTP, crystals of theKlenTaqM747K tertiary form were soaked in the fresh reservoir solution and after one hour the surrounding reservoir solution was exchanged. After a period of 14 h, dCMeTP or dCEtTP, was added in high stoichiometric excess: 1µl, 1.5µl and 2µl of 20 mM dCMeTP or dCEtTP per 4µl protein drop. Only crystals with dCMeTP could be used, while crystals with dCEtTP cracked and were destroyed. Data collection statistics of theKlenTaqM747K bound to DNA with one ddCTP incorporated and a dCMeTP in waiting position, are shown in Table 3.1. In Figure 3.11A, the experimental 2Fo-Fc map is shown in blue, where dCMeTP is Watson-Crick base paired to guanosine. 3.8-4.3 ˚A be-low the dCMeTP is Phe667, which interacts through aromaticπ-πinteractions with dCMeTP.
Opposite to the Phe667 is the adjacent dideoxycytosine base π-interacting with the dCMeTP.
The binding site of dCMeTP is also known as the insertion site.[158,161–164]In Figure 3.11B, the 4’-methyl group and the 3’-OH group of the dCMeTP are visible and the density was clearly visible at this position at the reasonably high resolution of 2.1 ˚A. At the top in Figure 3.11B Glu615 is shown, which is oriented to the methyl group and is 3.4 ˚A away. The dCMeTP is shown in Figure 3.11C where two Mg2+-ions are shown in lime spheres and motif C, which interacts with the Mg2+-ions, is shown at the bottom (arrow). To show the space occupied by motif C from Figure 3.11C, it is shown in surface representation in Fig. 3.11D. During dephosphorylation, which is associated with a translation and rotation of the dCMeTP to the post-insertion site (Fig. 3.12), the 4’-methyl group of the ribose from dCMeTP may come into the vicinity of motif C. In the insertion site is enough space for dCMeTP as shown in Figure 3.12. But extension of this modified triphosphate can be difficult, because there is very less space for the 4’-methyl group in the post-insertion site. To understand how such triphosphate analoga fit into theKlenTaqbinding pocket, further studies of this behavior as well as kinetical measurements are in progress in the AG Marx.
Figure 3.11: Structure ofKlenTaqM747K bound to DNA and dCMeTP are shown. A: Measured 2Fo-Fc map is shown atσ=1.8 in blue. The dCMeTP is Watson-Crick base paired to guanosine (G). Phe667 is labeled and interacts through aromaticπ-πinteraction with the dCMeTP.
The adjacent incorporated ddC is shown at the top. B: Density for the methyl- and the 3’-OH- group of the dCMeTP is clearly visible at this 2.1 A high resolution. From the top Glu615 is shown, which is in the direction of the methyl group (3.4 ˚˚ A away). C: Side view of the dCMeTP, two Mg2+-ions are shown as green spheres. Motif C is shown from the bottom. The post-insertion site is marked as “p.i.”. D: Same orientation as in C, but with a zoom to the dCMeTP and motif C. Motif C is shown in surface representation. During dephosphorylation, which is associated with a translation and rotation of the dCMeTP to the post-insertion site (p.i.), the 4’-methyl group of the ribose from dCMeTP may come into the vicinity of motif C.
Figure 3.12: Structure ofKlenTaqM747K bound to DNA and dCMeTP is shown. The methyl group of the Watson-Crick base paired dCMeTP has enough space in the insertion site.
4.1 Abstract
KlenTaqM747K, I614K was crystallized in ternary form with ddCTP and primer/template. The well ordered crystals diffracted to 1.7 ˚A. Until now, this is the highest resolution of the Klenow fragment of Thermus aquaticus DNA polymerase I (KlenTaq), as the wild-type structure was solved at 2.3 ˚A.
4.2 Introduction
Two single hydrophobic/cationic exchange mutants ofKlenTaqhave been found to possess en-hanced lesion bypass ability: M747K found at the Marx group and I614K from Patelet al.[165]. Isoleucine 614 is at a different position compared to methionine 747 and forms part of the hydrophobic pocket for binding the base and ribose portions of the incoming nucleotide. Klen-TaqI614K exhibits a 10-fold lower base misincorporation rate, as well as a high propensity to extend DNA mismatches. Furthermore, this mutant has lesion bypass activity on abasic sites (Fig. 1.5[1]) and vinyl chloride adduct ethenoA (Fig. 1.5[6])[165]. The error rate is more than 20-fold higher relative to the wild-type (80x10−5 forKlenTaqI614K compared to 3.3x10−5 for the KlenTaq wild-type) and the enzyme efficiently catalyzes both transition and transversion errors. Mutation I614K is a part of the highly conserved motif A (amino acids 605-617, Fig.
4.1) near motif C at the active center. The higher error rate and the lower fidelity combined with the lesion bypass activity makes this I614K mutant interesting to combine with M747K.
Figure 4.1: Motif A and Q-helix sequence alignment. Sequence alignment of 20 different prokaryotic organisms shows highly conserved sequences in motif A and in the Q-helix. ThepolAgenes were taken from GenBankTMand analyzed with NPS a(PBIL)[166]. In red are isoleucine (I614 forThermus aquaticus) from motif A and methionine (M747 forThermus aquaticus) from the Q-helix (adapted from[130]).
C. Gloecker from AG Marx, University Konstanz, combined these two mutants and analyzed the cumulative effects of this double mutantKlenTaqM747K, I614K. He found that the Klen-Taq M747K, I614K has a higher activity for the full length product by using damaged DNA (stabilized abasic site, 8-oxo-A or 8-oxo-G) compared to theKlenTaq M747K. Moreover, the KlenTaq M747K, I614K was able to amplify the full primer and add one additional base pair at the 3’-end independent of the template sequence. The incorporation of adenosine opposite an abasic site is known from the A-family polymerases as the A-rule.[167,168] In the case of the KlenTaqM747K, I614K this property was more pronounced than in theKlenTaqwild-type. Ad-ditional differences exist in the processivity where the distributiveKlenTaqwild-type shows an elongation of 10 nucleotides per binding (tested with DNA and polymerase blocking heparin).
By exchanging the hydrophobic methionine 747 to cationic lysine or arginine, the elongation extends to 21 nucleotides for the KlenTaq M747K and 37 nucleotides for an additional more cationicKlenTaqM747R mutant.[130]The cationic substitutedKlenTaqI614K, where the muta-tion is near the incorporated triphosphate shows a different behavior. It has the same distributive properties likeKlenTaqwild-type. So it can be assumed that only the cationic exchange at posi-tion 747 is responsible for the higher processivity.[130] Another difference between the variants lies in the thermostability. I614K has a destabilizing effect and shows a reduced half activity after 32 minutes at90◦C, while the wild-type and the M747K show half activity after 50 min-utes. In the case of M747K, I614K the half activity is increased again to 45 minmin-utes. So it can be assumed that cationic exchange at position 747 stabilizes the enzyme. Interestingly, the mutant M747R shows the highest thermostability with a half activity after 60 minutes at the same temperature.[130] The differences in behavior as well as the pre-steady state kinetics for
the second step is the elongation of this nucleotide[130]. From steady state measurements it is known that preferentially adenosine and less preferentially guanosine are incorporated opposite abasic sites[59]. Only minor differences in the KD for the incorporation of adenosine opposite abasic sites by theKlenTaq mutants were observed, but a dramatical increase in the turnover rate kpol (KlenTaqI614K 16-fold, KlenTaqM747K, I614K 20-fold). This leads to an increase in the efficiency up to 57-fold. Incorporation of dGTP opposite abasic sites was similar to the incorporation of dATP. However, the efficiency of incorporation was by a factor of 10-20 lower for dGTP as for dATP. This is caused by a lower turnover rate (kpol) and higher dissociation constant (KD)[130] (the results of kinetic measurements regarding abasic sites are summarized in the next chapter in table 5.1). To understand these differences and these cumulative effects from the double mutation, the structure of theKlenTaqM747K, I614K in complex with p/t and ddNTP was determined by X-ray crystallography.
wild-type M747K M747R I614K M747K/
I614K
distributivity 10nt 21 nt 37 nt 10 nt 21 nt
thermostability at90◦C 45min 50min 60min 32min 45min
kpol [s−1] dATP-dT 5.16±0.44 5.57±0.47 3.2±0.11 3.5±0.14 4.62±0.37 KD [µM] dATP-dT 15.3±4.7 11.3±3.7 6.84±1.09 2.2±0.03 1.46±0.35
relative efficiency# 1 1.5 1.4 4.7 9.4
Table 4.1: Summary of the differences in the mutants on the distributivity and thermostability, based on Gloeckners[130]results. KDis the dissociation constant for the binding of the triphosphate to the primer/template, measured by pre-steady state kinetics. kpolturnover rate was measured by pre-steady state kinetics.#Relative efficiency regarding the efficiencykpol/KDof the incorporation of dATP from the wild-type.
4.3 Material and Methods
4.3.1 Expression and Purification
E.coli BL21-Gold(DE3) harboring theKlenTaq M747K, I614K (double) mutant sequence in pASK-IBA37plus (IBA) with a N-terminal His6-tag and an ampicillin resistance were used for overexpression. Expression and purification of theKlenTaqM747K, I614K double mutant was done similar toKlenTaqM747K single mutant described in Chapter 3.3.2
4.3.2 Crystallization and data collection
The method used to obtain the ternary complex of theKlenTaqM747K, I614K was analogous to the strategy used previously for theKlenTaqM747K with DNA (Chapter 3.3.3) with the same primer/template:
Primer : 5’-GAC CAC GGC GC-3’
Template : 3’-CTG GTG CCG CGG GAA A-5’
+ ddCTP
The ternary complex was formed in a final concentration of 20 mM MgCl2 by mixing 3.0 mM DNA (p/t- complex) with 11 mg/ml protein and after a reaction time of 1h at room temperature, ddCTP was added in a molar ratio of 1:8.3:16.7 (protein:DNA:ddCTP). Crystals were grown using the vapor diffusion hanging drop method against a reservoir containing 0.1 M HEPES pH 7.1, 20 mM MnCl2, 0.1 M Na-acetate, 12 % (w/v) PEG 4000 at18◦C. Crystals appeared after 24h and grew in a hexagonal shape to the full size of 500 x 100 x 100 µm after 1 week (Fig. 4.2). The crystals were cryoprotected by soaking in reservoir solution containing glycerol in increasing concentrations (5 % steps to a final concentration of 20 %). Best datasets from
Figure 4.2: Crystal ofKlenTaqM747K, I614K with DNA and ddCTP.
APBS[160]. The Ramachandran statistic and outliers were detected using MolProbity[171,172].
4.4 Results and discussion
Digestion of 50µg protein with 1µg Factor Xa protease led to digested protein without His6-tag after 93 h at room temperature (lane 1-6 of Fig. 4.3 A+B). A lower protease concentration (lane 7-11 and lane 12-15) compared to the previously used, led, as expected, to more undigested protein in the same time. This is shown in Fig. 4.3 line 7-11 and 12-15, where in the western blot the anti-penta-His antibody is still bound to the undigested protein. After digestion of the His6-tag with Factor Xa protease, a Ni-column was used to separate digested from undigested protein and cleaved tag. Digested protein was used for gelfiltration and crystallization.
Figure 4.3: Digestion of the double mutant with different concentrations of Factor Xa protease. A: SDS PAGE, B:Western blot with anti-penta-His antibody: lane 1: Marker; lane 2-6: 50µg protein+ 1µg protease after 0 h, 17 h, 23 h, 44 h, 93 h; lane 7-11: 50µg protein + 0.5µg protease after 0 h, 17 h, 23 h, 44 h, 93 h; lane 12-15: 100µg protein + 0.5µg protease after 0 h, 17 h, 23 h, 44 h.
The space group of the crystals fromKlenTaqM747K, I614K was P3121 and cell axes were similar to those published for 3KTQ[124]. Therefore, it can be assumed, that the packing of the protein in the crystals is the same as those observed for the wild-typeKlenTaq. The difference between this structure and the 3KTQ wild-type structure is the mutation at M747K and I614K and the significantly higher resolution of 1.7 ˚A measured at the ESRF, ID23-1 compared to 2.3 ˚A for 3KTQ. So far, this is the highest resolution of the ternary complex of the Klenow fragment
from Taq DNA polymerase I. Density for the mutated residues was clearly observed at this resolution. Interestingly, there was an anomalous signal for only one of the two assumed Mg2+ -ions. At the wavelength of 1.072 ˚A, which was used for the measurements, the influence of Mg2+as anomalous scatterer is marginal, whereas Mn2+has a detectable anomalous signal (Fig.
4.4, 4.5). Therefore, it can be concluded, that Mn2+, which is also present in the crystallization
Figure 4.4: X-ray anomalous signal. Mn f’ and Mn f” (red) at the wavelength of 1.072 ˚A compared to Mg f’ and Mg f” (blue) (downloaded from http://skuld.bmsc.washington.edu/cgi-bin/edgeplots and modified).
setup and which is necessary for the crystallization, is bound to ddCTP. At the other metal ion position, near the 3’- position of the primer, no anomalous signal but spherical density was detected and it can be concluded that this position is occupied by one Mg2+-ion. Presumably, the same occupancy is in the wild-type structure as suggested by the B-factors for the Mg2+ -ion bound to the ddCTP in the wild-type structure (3KTQ) compared to the surrounding B-factors of the triphosphate atoms. It is known, that for error prone PCR, the polymerase is more inaccurate for the substrate selectivity in presence of Mn2+. Interestingly, only the position at the triphosphate is occupied by a Mn2+ while the other position, where the phosphorylation takes place, is occupied by a Mg2+. Hence, we have crystallized the protein in an error prone condition. The core root mean square deviation (rmsd) between 3KTQ and M747K, I614K is 0.31 ˚A, which indicates that the two are very similar.
Figure 4.5: X-ray anomalous signal at the position of the assumed Mg2+, which indicates (compared to the second ion (white cross)) that an anomalous scatterer is present. The position is likely occupied by Mn2+, which is essential for crystallization in this condition.
Data collection
KlenTaqM747K, I614K bound to DNA and ddCTP
Beamline ESRF ID 23-1
Cell dimensions a,b,c [ ˚A] 108.6, 108.6, 90.6 Cell dimensionsα, β, γ [ ˚ ] 90.0, 90.0, 120
Rcryst[%] 16.7
Rf ree[%] 19.8
r.m.s. deviation bond length [ ˚A] 0.006 r.m.s. deviation bond angle [ ˚A] 0.932 Average B factors [ ˚A2] 23.6 Max. likelihood estimate for coordinate er-ror [ ˚A]
0.19
Table 4.2: Data collection statistics forKlenTaqM747K, I614K in ternary complex with DNA and ddCTP.#3 Datasets were merged with XSCALE. Rmeas= redundancy independent R-factor (intensities) (For definition of Rmeassee Diederichs & Karplus (1997), Nature Struct.
Biol. 4, 269-275)∗Values in parentheses correspond to those in the outer resolution shell.