Interactions of non-polar and click-able nucleotides in the confines of a DNA polymerase active site

Article abstract of DOI:10.1039/c2cc34181f

Modified nucleotides play a paramount role in many cutting-edge biomolecular techniques. The present structural study highlights the plasticity and flexibility of the active site of a DNA polymerase while incorporating non-polar Click-able nucleotide analogs and emphasizes new insights into rational design guidelines for modified nucleotides.

Full text of DOI:10.1039/c2cc34181f

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Interactions of Non-Polar and “Click-able” Nucleotides in the Confines
of a DNA Polymerase
Samra Obeid,*a Holger Bußkamp,a Wolfgang Welte,b Kay Diederichsb and Andreas Marxa
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
Modified nucleotides play a paramount role in many cutting-
edge biomolecular techniques. The present structural study
highlights the plasticity and flexibility of the active site of a
DNA polymerase while incorporating non-polar “Click-able”
10 nucleotide analogs and emphasizing new insights into rational
design guidelines for modified nucleotides.
Regarding biological core technologies such as next-
generation sequencing1, single molecule sequencing2,
microarray analysis based on labeled DNA amplificates3,
15 DNA conjugation4, or the in vitro selection of ligands like
aptamers by SELEX (systematic enrichment of ligands by
exponential
amplification)5,
functionalized
2’-
deoxynucleoside triphosphates (dNTPs) play a fundamental
role. One approach to improve the incorporation efficiency of
20 modified substrates is to vary the linkage connecting
nucleotide moiety and modification of choice. In most cases
the modifications were introduced at the nucleobase, in
particular, in case of pyrimidines at C5 position1-6. For
rational linkage design the knowledge of the structure-
25 function relationship of the enzyme is of great importance.
Two recently solved structures showed KlenTaq (the large
fragment of Thermus aquaticus DNA polymerase I) caught
processing several modified dNTPs7. These studies focused on
flexible and polar modifications that have distinct hydrogen-
30 bonding capability. It turned out that the polar functionalities
allow proper substrate-protein interaction and thereby might
improve the substrate properties. However, little is known
about rigid and nonpolar modification. So far, only one DNA
Fig. 1 a) Structure of dT*TP and primer extension. A representative
PAGE analysis of the competition experiment dT*TP vs dTTP using
KlenTaq is shown. The ratio of dT*TP/dTTP was varied from 1/1 to
100/1 (1/1, 2/1, 4/1, 10/1, 20/1, 50/1, 100/1). b) same as in a) for dC*TP.
c) Primer extension using dC*TP followed by Click reaction. Partial
sequence of the employed primer/template construct is indicated on the
right. Lane P: primer only; lane 1: primer extension by KlenTaq in
presence of dATP, dGTP and dTTP; lane 2: all four natural dNTPs; lane
3: same as lane 2 followed by incubation with streptavidin (STV); lane
4: same as in lane 1, but in the presence of dC*TP; lane 5: same as in
lane 4 followed by incubation with STV; lane 6: same as in lane 4
followed by Click reaction with azido-modified biotin; lane 7: same as
line 6 followed by incubation with STV; lane 8: same as lane 4 followed
by incubation with azido-modified biotin (structure is shown) without
BTTAA and CuSO4; lane 9: same as in lane 8 followed by incubation
with STV. For experimental detail see ESI †.
polymerase structure capturing
a
rigid and nonpolar
35 modification is available7a. This study suggested that the rigid
and non-polar modification results in the distortion of the
interaction between the enzyme and the substrate that leads to
a Department of Chemistry and Konstanz Research School Chemical
40 Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz,
Germany. Fax: (+49) 7531-88-5140; Tel: (+49) 7531-88-5139; E-mail:
andreas.marx@uni-konstanz.de
b Department of Biology, University of Konstanz, Universitätsstrasse 10,
45 7†8E45le7ctKroonnisctanzS,uGpeprlmemaneyn.tary Information (ESI) available: See
DOI: 10.1039/b000000x/
‡ Data deposition: The atomic coordinates and structure factors have
been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes
4ELT, 4ELU and 4ELV).
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the observed low incorporation efficiency. However, in some
cases rigid linkers are needed to enable e.g. spectroscopic
of these experiments revealed that KlenTaq incorporates
dT*MP and dC*MP with only 7- and 2-fold lower efficiency
properties. Therefore, we extended the functional and 35 compared to the natural counterparts, respectively (Fig. S1).
structural studies with a new set of rigid C5 modified
nucleotides (Fig. 1). The introduction of an aromatic ring in
the rigid linkage should enable interactions to the protein.
Further, these modified nucleotides serve as precursors, which
Encouraged by the excellent acceptance of the modified
substrates, we next investigated the access of the terminal
alkyne group via Click reaction (Fig. 1c, S2 and Scheme S2).
First, we performed primer extension experiments using
5
can be post-synthetically addressed by Cu(I)-catalyzed azide- 40 modified substrates to introduce a terminal alkyne group into
alkyne cycloaddition (“Click” chemistry), due to the terminal
DNA (Fig. 1c, lane 4). The mono alkyne functionalized DNA
product was subsequently used for the Click reaction. An
azido-modified biotin was clicked to the modified DNA. After
4h reaction time a quantitative conversion to the biotin labeled
10 alkyne function6l, 8. We present crystal structures showing
KlenTaq caught during the incorporation process of modified
nucleotides. The structures reveal that the interaction pattern
of the aromatic ring with the protein might account for the 45 DNA product is observed, which shows a slight decrease in
efficient processing of the analogs by KlenTaq.
retention time, due to the additional bulk of the introduced
modification (Fig. 1c, lane 6). Further, incubation with
streptavidin and the resulting change in the electrophoretic
mobility revealed that the Click reaction product indeed
15 Starting from 5-iodo 2’-deoxyuridine the modification was
introduced by palladium-catalyzed cross-coupling6n,
with
9
1,4-dienthynylbenzene. Next the modified thymidine analogs
were transformed to the corresponding cytidine analog10, 50 carried biotin (Fig. 1c, lane 7).
followed by the conversion into the respective thymidine- or
20 cytidine triphosphate analogs11 (for details see ESI †).
Having the modified pyrimidine analogs in hand their
acceptance and incorporation efficiency compared to the
To gain structural insight into how the modified pyrimidine
analogs are processed by KlenTaq we crystallized KlenTaq
bound to a primer/template complex and the modified dT*TP
and dC*TP positioned at the dNTP binding site
natural substrates by KlenTaq were investigated by 55 (KlenTaq(dT*) or KlenTaq(dC*)). To stall the respective
competition experiments3c, 7(Fig. 1a,b). In these single
25 nucleotide incorporation experiments the modified substrates
directly compete with the natural counterpart for
incorporation. The objective was to determine the relative
modified substrates in the position poised for catalysis the
polymerization reaction was terminated by the incorporation
of ddCMP or ddGMP at the 3’-primer terminus. The
structures were solved by difference Fourier techniques and
ratio of modified versus natural substrate that results in equal 60 provide snapshots of the incorporation of the modified
incorporation efficiencies. This ratio was assigned using 20%
30 denaturing polyacrylamide gel electrophoresis exploiting the
different retention times of the reaction products, due to the
additional bulk of the modification6n, 7a, 12. The quantification
triphosphates at resolutions of 2.2 Å and 1.8 Å, respectively
(Table S1 and Fig. 2, S3 and S4).
In KlenTaq(dT*), the polymerase adopts a similar overall
structure as it is observed for KlenTaq in complex with DNA
Fig. 2 KlenTaq(dT*) structure (gray). a) Zoom into the active site of KlenTaq(dT*). The incoming dT*TP is stabilized via Watson Crick base pairing.
The O helix packs against the nascent base pair. R660 is indicated as stick. b) Interaction pattern of the modified dT*TP with the protein side chains
R587 and K663 (red dashed lines indicate cation-π interactions). Distance of the α-phosphate to the 3’-primer terminus is shown in blue dashed line.
Gray dashed lines indicate further interaction pattern. c) Close-up view of the complexation of Mg2+ ions by the incoming dT*TP and the amino acids
responsible for catalysis. d) – f) Shown are the same cut-outs and orientations as in a) – c) for KlenTaq(dT*) (gray) superimposed with
KlenTaq(1QTM) (green). All distances are in A
.
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chains like arginine or lysine. This fact might explain the
and ddTTP (PDB: 1QTM; KlenTaq(1QTM)), resulting in a
low r.m.s.d. for Cα of 0.417
KlenTaq(1QTM) the recognition of dT*TP relies on Watson–
Crick base pairing and π-stacking interaction to the primer
strand. Further all components required for efficient catalysis
are properly arranged, except for two amino acids Arg660 and
A
.
In analogy to 60 efficient processing of dT*TP and dC*TP. For instance, the
earlier employed thymidine anlog7a that bears a rigid non-
polar modification lacking an aromatic ring is less effectively
processed as dT*TP (i.e. 7- versus 2500-fold reduction of
incorporation efficiency compared to dTTP). In line with this
5
Arg587. Arg660 is positioned to make room for the rigid 65 findings, Burgess and coworkers demonstrated that dyes
modification, thereby disabeling interactions with the 3’-
primer terminus, which are observed in the unmodified case
attached to the nucleobase via a rigid conjugate linkage show
enhanced spectroscopic properties and their substrate
properities increase with the linker bearing an aromatic ring14.
These insights and the present study suggest that
10 (Fig. 2a,d). Similar effects were observed earlier in case of
another rigid non-polar modification that is processed about
2500-fold less effective by KlenTaq7a. However, in the present 70 implementing an aromatic ring at the discussed position in
study the incoming dT*TP is further stabilized by Arg587.
The guanidinium group of Arg587 is located above the
15 aromatic ring of the modification allowing cation-π
interaction (Fig. 2b). Further Arg587 stabilizes the phosphate
modified dNTPs may improve their substrate properties. This
enhancement of design guidelines for the development of new
modified dNTPs in combination with directed evolution of
DNA polymerases15 will stimulate the development of future
backbone of the 3’-primer terminus. In addition, Lys663 is 75 applications.
located on the other side of the aromatic ring also forming
cation-π interactions. Thus the aromatic ring is sandwiched
20 between two positively charged amino acid side chains
(Arg587 and Lys663). While the C5 modification at the
nucleobase does not induce a conformational change of
Lys663, it clearly affects the conformation of Arg587 (Fig.
2e). The distance from the primer 3′-terminus to the α-
25 phosphate of dT*TP is similar to the one observed in
KlenTaq(1QTM) underpinning that dT*TP is properly aligned
for catalysis (Fig. 2c,f). This interaction pattern may account
for the proficient processing of dT*TP by KlenTaq.
Notes and references
1
(a) D. R. Bentley, et al., Nature 2008, 456, 53; (b) J. Bowers, et al.,
Nat. Methods 2009, 6, 593.
(a) H. Ruparel, L. Bi, Z. Li, X. Bai, D. H. Kim, N. J. Turro, J. Ju,
80 2 Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5932; (b) T. D. Harris, et
al., Science 2008, 320, 106.
3
(a) J. Guo, et al., Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9145; (b)
J. R. Pollack, C. M. Perou, A. A. Alizadeh, M. B. Eisen, A.
Pergamenschikov, C. F. Williams, S. S. Jeffrey, D. Botstein, P. O.
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Marx, Angew. Chem. Int. Ed. 2012, 51, 254.
85
90
95
4
5
C. M. Perou, et al., Nature 2000, 406, 747.
The obtained structure of KlenTaq capturing dC*TP
30 (KlenTaq(dC*)) shows very similar features as observed for
KlenTaq(dT*) highlighted by the superimposed structures
(Fig. S4). Once more, the incoming dC*TP is properly aligned
and interacts via Watson-Crick base pairing with the
templating G. In analogy to KlenTaq(dT*), dC*TP is
35 additionally stabilized by cation-π interaction with Arg587
(Fig. S4b). The assembly of the catalytic residues with the
required metal ions are very similar to KlenTaq(dT*) and
consequently very alike KlenTaq(1QTM) indicating that
dC*TP is properly positioned (Fig. S4c).
T. S. Seo, X. Bai, D. H. Kim, Q. Meng, S. Shi, H. Ruparel, Z. Li, N.
J. Turro, J. Ju, Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5926.
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6
In summary, the obtained structures of KlenTaq capturing100
modified substrates in the active site illustrate the plasticity of
the enzyme and highlights the importance of the flexible
40
regions of the dNTP binding pocket defined in the model of
“active site tightness”13. Attaching the modification at the C5105
45 atom of the pyrimidines has two major advantages. Firstly,
modifications at this position cause minor disruption of the
7
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DNA duplex, since it points to the major groove. Secondly, it
also results in minor disruption of the enzyme’s ternary110
complex, since KlenTaq possesses sufficient plasticity to
50 adapt to the structure of the modified substrates. This fact
underpins the idea that the enzyme is able to respond to the
8
9
modified115 1110 T. Kovács, L. Ötvös, Tetrahedron Lett. 1988, 29, 4525.
J. Gaster, A. Marx, Chemistry 2005, 11, 1861.
incoming nucleotide by interacting with the
12 A. Baccaro, A. Marx, Chem.--Eur. J. 2010, 16, 218.
substrates. Previous structural studies of flexible and polar
modified substrates revealed hydrogen-bonding capability
55 might improve their substrate properties7. The present study
13 E. T. Kool, Annu. Rev. Biochem. 2002, 71, 191.
14 L. H. Thoresen, G.-S. Jiao, W. C. Haaland, M. L. Metzker, K.
Burgess, Chem.--Eur. J. 2003, 9, 4603.
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introduction of an aromatic ring enables new interactions as
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cation-π interaction to positively charged amino acid side
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