N-Methyl Mesoporphyrin IX
Bound to Human Telomeric G-Quadruplex DNA
Mason McCool '17, Cole Meier '19
Contents:
I. Introduction
Guanine rich DNA sequences have the ability to form tertiary
structures, called G-quadruplexes
(GQs).
Their related
consensus sequences are found in various organisms,
greatly conserved in mammals, but present less
frequenently in lower organism such as bacteria.1 GQs
are four stranded DNA sequences, differing from the normal duplex
DNA structure, and can form inter or intra molecularly.
These structures can form at various parts of the human genome, such
as gene promoters, replication origins, and intron/exon borders.
However, GQs are most
prevalently found within telomeres,
where there are up to 10,000 bases of TTAGGG repeats, that are
able to readily form these structures. They form at the 3'
overhang, which is a single stranded portion allowing for
the formation of intramolecular GQs,
dGGG(TTAGGG)
3.2
One major importance of 3'
overhang GQs
in-vivo can be attributed to their end capping that protects chromosome ends.
Because of their extensive four stranded
bulky structure, they provide resistance against nucleases. Along
these same lines, it also has the ability to inhibit telomerase
(the enzyme that adds nucleotides to extend the telomere
when it is shortened due to DNA replication cycles), thus regulating it's
activity and relative telomere
length. This aspect of GQs
has been taken advantage as a target for anti-cancer drug
therapy.3 There are ligands
that can bind and stabilize GQs,
leading to greater telomerase4
inhibition. If a significant amount of inhibition occurs, this will eventually lead to telomere
shortening and eventually cell senescence or apoptosis.3
There have only been a moderate amount of these structures
determined for different human telomere
GQ-ligand
complexes. Many of these are low resolution, so
specific features of ligand-GQ
interactions cannot be determined. It is also noteworth that
no highly selective GQ
ligands have been
studied, until the
study of N-methyl
mesoporphyrin IX (). Porphyrins
contain a conjugated macrocyclic structure and can have various
substituents. NMM most notably has two carboxylate groups on the exterior
portion of the molecule and an N-methyl group that protrudes down
away from the aromatic plane in the center of the molecule.6
NMM was
originally studied as an inhibitor of ferrochelatase, the enzyme
that catalyzes the insertion of iron for heme biosynthesis.7 However, now it is of
interest because of its selectivity for parallel GQs
unlike other anti-cancer GQ
ligands studied
previously.
II. G-Quadruplex Structure
GQs structures
form from guanines held together by Hoogsteen
base-pairing between four guanines forming a tetrad structure. There
is hydrogen bonding between
as well as
of two separate
guanines, forming 8 hydrogen bonds total per
.
Then the three tetrads stack upon each other due to
stabilizing
between the
aromatic groups on guanine, similar to the alpha
helical base stacking that stabilizes the general DNA
secondary structure.
Three
guanines that occur in sequence are stacked upon each other. Each of
the guanine sequences are connected through three base loops (TTA),
occuring three times to connect all four guanine sequences and forming
three tetrads
. These structures are further stabilized in physiological buffer
conditions by monovalent cations such as Na+
and K+
that interact with the carbonyl oxygen on the guanines. There are 3
cations present per GQ
structure, one for each tetrad, that stabilize via charge screening.5
While the general GQ
structure is outlined by guanine base pairing and cation
interactions, there can be different alignments of the sequence or
sequences of nucleotides that result in a specific type of GQ
structure. When all three guanine base regions are oriented in the
same directionality (upward/downward relatively) it is considered
parallel, while if they are not it is antiparallel.5
As mentioned prior, NMM
prefers the parrallel conformation of GQs
. It can even induce a conformational change to the parallel GQ structure,
unique compared to other ligands.
Knowing the structure of GQs
alone, helps determine possible ligands
that can be used to further stabilize the GQ
and be used as anti-cancer drugs.
III. G-Quadruplex Dimer Formation, Not Indicative
of Native State
When in a crystal structure the telomeric
DNA GQs form in dimers.
The 5’ face of both of the GQs
form a dimer through pi stacking interactions and a bridging K+
ion. In addition, there is reverse base pairing between A1 of one
DNA GQ strand and T12
of the other DNA GQ strand
.5 There can even be some interactions between multiple dimers. However, dimer formation is
not biologically relevant; GQs do not
interact amongst each other in vivo.
Dimer formation is attributed to the much higher
concentration of DNA and packing forces experienced in a crystal
structure. This leads to the a diminished area of the GQ
being exposed for interactions with K+
present in the solution, not indicative of the state in the cell.
The GQ DNA
when bound to NMM
is monomeric in solution, a representative state of DNA in-vivo.
Therefore, the dimer interactions do not attribute to the
stability of GQ
structure and only arise through experiment conditions required
for x-ray crystallography. Fortunately, due to comparisons between
other GQ monomeric
structures, the dimerization does not significantly change the
interactions that are relevant to directly compare this study to the structure in vivo. The
formation of the dimer still results in a 1:1 stoichiometry
between NMM and GQs
in solution, not affecting the information to be gained from
analyzing these interactions.5 It is important to focus
on the NMM-GQ
interactions specifically and not the dimer interactions when
studying the ligand
effects on stability as a possible telomerase
inhibitor anti-cancer drug.
IV. N-methyl mesoporphyrin IX Interactions
One molecule of NMM
binds to the 3’ face of the GQ,
similar to other anti-cancer ligands that have been analyzed
binding to the same GQ
DNA sequence. This 3’ face is made available for binding in the
parallel GQ
conformation, while antiparallel structures have loops that block both
the 3’ and 5’ tetrads from ligand binding.5 The
macrocycle structure, the inner portion of the ligand, binds to
the accessible 3’ tetrad through pi stacking interactions.
The portion of NMM is 3.6 angstroms away from the tetrad, which is
further than other ligands and indicates that there must be another
reason for GQ
selectivity.
Another interaction is the N-methyl group of NMM
that is bent 44.8 degrees away from the aromatic plane of the
ligand towards the K+
ion channel. This causes the ligand core to be off
center and asymmetrically bind to the GQ.5
However this could lead to positive interactions between the nitrogens in the ring of NMM and
the K+,
or it could also benefit side chain interactions with the
telomeric DNA grooves. These core
interactions and their importance in binding are
emphasized by the decreased movement of the core NMM
atoms and the increased disorder of the peripheral atoms as
indicated by the heat map
HeatmapKey
.
In addition to these core interactions, the exterior portion
of the ligand also interacts in a stereo-specific manner with the GQ
structure. The two propinate interact with the
. The first
interacts through water mediated hydrogen bonding with
straddling G3 backbone phosphates.8 The second also interacts
via water mediated hydrogen bonding with on the 3' sugar of G22.8
There are no electrostatic attractions between the GQ
and NMM. This
lessens the strength of binding, with a binding constant of ~105 M-1.5 Although the binding constant is unexceptional, the
specificity for parallel GQs
is the noteworthy attribute of NMM
as an anti-cancer ligand.
This specificity arises from the structural adjustment of NMM
in order to align with the 3’ tetrad along with the isomerization
of the GQ to adopt
the parallel conformation. At this point, NMM
is a starting point for research towards more selective GQ
binding ligands.5 These ligands
will lead to anti-cancer drugs with better efficacy towards
inhibiting telomerase. Tthe specific GQ
binding, will limit side effects from non-specific binding of other
molecules or other DNA structures.
V. References
1) Rhodes, Daniela, and Hans J. Lipps. "G-quadruplexes and Their Regulatory Roles in Biology." Nucleic Acids Research 43.18 (2015): 8627-637. Web.
http://nar.oxfordjournals.org/content/early/2015/09/07/nar.gkv862.full.pdf+html>