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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5319_Библиотеки_им_академика_М_И_Перельмана.pdf
. • The Filling of Hydrophobic Pockets and Interference with aWater Network
. Fig. 21.10 From the 6-amino-quinazolinone scaffold 21.12, the
listed derivatives could be synthesized and tested by adding different
substituentsR (red, upper left). To our surprise, even the best compounds of this series remained in the single-digit micromolar range.
As an alternative inhibitor scaffold, lin-benzoguanines 21.13 were pro-
vided with hydrophobic residues in the 4-position (lower left). Despite
very good inhibition of the basic scaffold, the substituted derivatives
failed to achieve a signicant improvement in afnity. The crystal
structure with 21.14 shows (right) that the indicated phenylethyl sub-
look at individual derivatives. It was surprising that the
small backbones without any side chains already showed
single-digit micromolar binding. Adding another small
substituent to the hydrophobic pocket initially led to
aloss of binding afnity. This loss of afnity could only
be compensated for by lling the hydrophobic pocket
with an aromatic residue. Acomparison of the arrangement of the water molecules in the different inhibitor
structures was revealing. In the unsubstituted derivatives, several water molecules form anetwork between
the two presumably charged aspartate residues 102 and
280 (. Fig.21.11, left). This network signicantly contributes to the residual solvation of these two polar acid
residues in the protein. Presumably, the water molecules
in this region buffer the accumulation of negative charges
on the two adjacent acid groups. All of the derivatives
listed in . Fig.21.10 span this region of the water net-
work with ahydrophobic linker in order to place their hydrophobic substituents in the small hydrophobic pocket
stituent binds into asmall hydrophobic pocket of Val45, Leu 68, and
Asn70. The ligand-bound structure is shown with orange carbon atoms and the ligand-free structure is displayed with gray carbon atoms.
(7 https://sn.pub/DNgZHZ)
at the end. In doing so, however, they inevitably destroy
the water network. This has its price!
An afnity comparison between compounds 21.15
and 21.16 (. Fig. 21.11) was striking. The derivative
with a 7-dimethylamino group on the quinazolinone
scaffold 21.15 lost binding afnity by afactor of more
than10 compared to the unsubstituted derivatives. Replacing one of the methyl groups with abenzyl group
(21.16) partially restores the lost afnity. The crystal
structure of this derivative shows that the benzyl group
is not oriented towards the small hydrophobic pocket,
but rather towards apocket occupied by uracil33 in the
natural substrate (. Fig.21.2, green pocket). With this
result, anew concept for further design was obvious. Under no circumstances should the water network between
Asp 102 and Asp 280 be crossed by ahydrophobic linker.
Furthermore, ahydrophobic group should be added facing the uracil33 pocket to allow the ligand scaffold to
grow into this pocket.
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
21
. Fig. 21.11 Left The backbone of lin-benzoguanine 21.13 binds to
the protein with 4.1 μM, leaving the water network (red spheres) between the two presumably negatively charged aspartates 102 and 280
intact. Right The binding afnity of the quinazolinone derivative with
a7-dimethylamino group (21.15) decreases by afactor of10 compared
with the unsubstituted derivative. However, when one of the two meth-
21.9 With aSalt Bridge:
Finally Nanomolar!
Synthetically, the desired modications of the substituent were easier to achieve on the lin-benzoguanine parent
scaffold. Unsubstituted lin-benzoguanine 21.13 displays
awater network containing ve distinct water molecules
in the crystal structure with the enzyme (. Fig.21.11,
left). It was decided not to change this network in the
next design cycles, so that derivatization rst concentrated on the 2-position and the study of the 4-position
was postponed (see below). The sole attachment of
amethyl group to the 2-position (21.17) improves afnity
by afactor of2.7 (. Fig.21.12).
If the methyl group is then exchanged for an amino
(21.18) or methylamino group (21.21), the binding con-
stant will dramatically improve into the two-digit nanomolar range. The introduction of an amino group in the
2-position of the lin-benzoguanine scaffold improves the
afnity by afactor of50! How can this surprising result
be explained? The hydrogen bond to the carbonyl group
of the main chain in Leu 231 was discussed in Sect.21.5.
This functional group is part of the peptide bond, which
can ip its orientation like aswitch. The lin-benzogua-
nine scaffold also forms ahydrogen bond to this carbonyl
group of Leu 231, and the introduction of an amino group
in the 2-position transforms the imidazole portion into
aguanidine-like moiety (. Fig.21.13, center, red). Such
achange increases the basicity of the scaffold and possibly
alters its protonation. However, this is not the only guanidine-like group in the molecule. The aminopyrimidone
moiety also contains such agroup (. Fig.21.13, center,
blue). Measurements of pKa values in aqueous solution
yl groups is replaced by abenzyl group (21.16), an increase in activity
is again obtained. The crystal structure with this derivative showed
that the benzyl group is not oriented towards the small hydrophobic
pocket (blue pocket in . Fig. 21.2), but projects into the uracil 33
pocket (green pocket in . Fig.21.2)
indicate that the aminoimidazole is the group with the
higher pKa value, so this group should be protonated rst.
This was experimentally veried by Manuel Neeb. As described in Sect.4.4, isothermal titration calorimetry can
be used to determine how many protons are transferred
during ligand binding to the protein. The titration was
carried out at pH7.8. In total, 21.18 takes up one proton
when it binds to the protein. But where does the proton
go? In 21.17, the guanidine moiety in the imidazole ring
is missing; nevertheless, the ligand still takes up aproton
(. Fig.21.13, left). In contrast, no proton is taken up
when the guanidine group in the pyrimidone moiety is
removed (21.20). These results suggest that the proton is
taken up by the aminopyrimidone moiety, even though
the pKa of this group is lower by 1.3pH units! At rst
sight, this seems to be very contradictory. To be sure, we
replaced the two charged aspartates 102 and 156 in the
binding pocket of the TGT with uncharged asparagines
by mutagenesis (. Fig.21.13, right). Interestingly, this
eliminates the uptake of aproton by 21.21, again emphasizing that the effect occurs at the aminopyrimidone
moiety. The observed protonation step is induced by the
two adjacent and negatively charged Asp residues, which
locally shifts the pKa values quite strongly.
However, this does not explain the sudden increase
in afnity due to the introduction of an amino group on
the lin-benzoguanine scaffold in the 2-position. This observation would have been easily explained by the more
basic nature of the aminoimidazole moiety, which would
have resulted in acharge-assisted hydrogen bond to the
carbonyl group of Leu 231. Further structural studies
were performed at different pH values to rule out the formation of this charge-assisted interaction. Finally, any
. • With aSalt Bridge: Finally Nanomolar!
. Fig. 21.12 Substitution of the lin-benzoguanine parent scaffold
21.13 in the 2-position leads to asignicant improvement in binding
afnity. In particular, the introduction of a 2-amino group (21.13
⇨ 21.18) leads to a tremendous increase in binding afnity. Larger
substituents are accommodated by the U33 pocket, but are mostly
disordered there. The morpholino derivative 21.22 represents asingle-digit nanomolar inhibitor. The attempt to introduce another afnity-increasing salt bridge via 4.11 to 4.12 has no effect, since the salt
bridge remains exposed to the solvent (cf. Sect.4.8, . Fig.4.13)
. Fig. 21.13 The parent scaffold of the lin-benzoguanine derivative
21.21 (center) has two guanidine-like groups that account for the ba-
sic character of the compound. The aminoimidazole moiety (red) has
apKa of5.7 in water, while the aminopydimidone moiety (blue) has
apKa of4.4. Therefore, protonation should occur more readily at the
imidazole moiety. ITC measurements (left) show the uptake of one
proton upon binding to the wild-type TGT. For 21.17 (top left), which
cannot accept aproton at the imidazole moiety, this still results in the
uptake of one proton upon protein binding. In contrast, no proton
is taken up upon binding of 21.20 (bottom, left). This suggests that
despite the lower pKa, the aminopyridmidone moiety is protonated.
Mutagenesis and crystal structure determination conrm these data
(right). Only the binding of 21.21 to the wild type, in which two negative charges occur in close proximity at Asp 102 and Asp 156, shows
proton uptake. If Asp is replaced by Asn by mutagenesis, one negative
charge on each side of the protein variants is lost, and 21.21 binds to
the protein without picking up aproton
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
additional protonation effects on the side of the protein
that could have disturbed the found protonation inventory could be ruled out. Thus, the observed increase in
afnity could only be explained by the absence of repulsive
effects in the formation of the two parallel hydrogen bonds
(. Fig.21.14). If the donor or acceptor groups are on the
same side of the binding partners that come together to
form the complex, no additional repulsive effects between
the hydrogen atoms in the twinned H-bonds need to be
overcome during complex formation (. Fig.21.14, left).
The repulsive effects are also present there, but they had
to be overcome already during the synthesis of the individual components. Therefore, they are not important for
the balance of complex formation. If, on the other hand,
donor and acceptor groups arrange in alternate fashion
on the partners, the repulsive effects occur during complex formation. Since these effects come at acost during
complex formation, they will weaken the strength of the
H-bonds that are formed. This effect is surprisingly strong
and it has been described for the rst time in host–guest
. Fig. 21.14 When two binding partners come together to form
two parallel hydrogen bonds, it is crucial for the contribution to the
strength of these H-bonds whether the two donors and acceptors
are on the same side (left) or on opposite sides (right) of the binding
partners. Left There are no repulsive interactions of the H-atoms to
overcome during complex formation. Any repulsive effects had to be
exceeded during the synthesis of the binding partners and, therefore,
do not have to be paid for during complex formation. Right These
repulsive effects arise during complex formation once the H-bonds are
formed. This weakens the afnity contribution to be achieved during
complex formation
complexes. Interestingly, such effects also occur during
H-bond formation between the nucleobases on the individual steps of DNA (Sect.14.9). For the optimization
of our TGT inhibitors, this was of course an extremely
welcome additional afnity contribution!
It has already been demonstrated that lling the uracil 33-binding pocket is associated with an improvement
in the afnity. Therefore, groups were introduced onto
the 2-amino group. However, a methylene group was
used as alinker to keep the amino group electronically
unconjugated to the added aromatic substituents. Of
the synthesized derivatives, morpholine derivative 21.22
proved to be the strongest binder. It also has the best water solubility. Interestingly the added side chains in this
area are not clearly visible in the electron density. They
are probably in adisordered state in the binding pocket
(. Fig.21.17, left). This speaks against agood enthalpic
interaction for these groups in this area, but this effect
should be compensated for due to entropic reasons so
that agood contribution to the free energy is achieved
in the sum, and overall the binding afnity is improved.
This situation is explained in an example in Sect.4.10.
After optimization of the substituents at the 2-position, an enlargement of the molecular scaffold of the
lin-benzoguanines at the 4-position was approached.
Special attention was paid to the perturbation of the polar
interactions with the water network. This network buffers
the charges between Asp 102 and Asp 280 (. Fig.21.11,
left). Purely hydrophobic side chains had not yielded the
desired increase in afnity (. Fig.21.10). Therefore,
abasic nitrogen atom was introduced into the linker
to actively participate in the water network. As aresult,
single-digit nanomolar inhibitors have also been successfully developed (. Fig.21.15). Crystal structures
21.23 and 21.24 (. Fig.21.16, top) show that the water
network is successfully incorporated and stabilized. The
introduced polar nitrogen probably binds to Asp 280 in
protonated form via asalt bridge. It also participates in
the water network.
However, another phenomenon was observed, the
signicance of which only became clear as the project
progressed. The two inhibitors differ by only one mem-
21
. Fig. 21.15 Substitution of the lin-benzoguanine parent scaffold
21.21 in the 4-position by abasic nitrogen in the side chain leads to
asignicant improvement of binding afnity down to the nanomolar
range. Terminal cycloaliphatic groups proved to be the best representatives of the series (21.23, 21.24)
ab
cd
. • With aSalt Bridge: Finally Nanomolar!
. Fig. 21.16 The crystal structures with the 4-substituted lin-benzo-
guanine derivatives show that the basic nitrogen in the chain is incorporated into an H-bonding network with the water cluster and with
Asp 280 (top). At rst glance, 21.23 and 21.24 appear to adopt an
almost identical binding mode. On closer inspection, however, there is
acrucial difference that, as was shown later, is of great importance for
the stability of the protein (bottom). The cyclohexyl ring (blue) in 21.24
the entire loop with the attached helix, which becomes disordered at
the end. It can no longer be seen in an orderly fashion in the crystal
structure (in the blue structure, this part is missing in the region of the
ellipse, whereas it is clearly visible in the ochre structure). As will be
shown later, this breakdown of the ordered structure of the loop–helix
motif has amassive inuence on the dimer stability of the enzyme.
(7 https://sn.pub/eM2mQk)
21.23, displacing Val45 from its position in the uncomplexed protein
(see red arrows). The cyclopentyl ring in 21.23 is slightly smaller, so it
does not cause this displacement. When Val45 is dislocated, it continues like aseries of dominoes, rst on Thr47 (red arrows) and then on
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
21
. Fig. 21.17 The crystal structure of morpholino derivative 21.22
with asubstituent in the 2-position (left) shows no well-dened difference electron density (green mesh) around the morpholino side
chain in the region of the uracil33 pocket (green arrow). This obser-
vation suggests astrong disorder of this substituent scattered across
multiple spatial orientations. MD simulations conrm this hypothesis
and indicate two possible placements of the side chain. By incorporating abasic nitrogen atom into the side chain at the 4-position of
the lin-benzoguanine scaffold in 21.24, the hydrogen-bond network
ber in the terminal cycloalkyl substituent, a ve- or
six-membered ring. The slightly smaller ve-membered
ring ts easily into the ribose-34 pocket (blue pocket, see
. Fig.21.2, bottom), leaving the protein virtually un-
changed from the uncomplexed structure. In contrast,
the six-membered ring requires more space and exerts
steric pressure on the side chain of Val45 (. Fig.23.16,
bottom, red arrows). This triggers adomino-like cascade
of displacements. As aresult, the following residues (see
Thr47) also shift their positions, and nally the entire
loop–helix motif from sequence position 45 to 63 is
disordered. Structurally, this section of the protein collapses. Much later, we realized that this loop–helix motif
is crucial for the stability of the enzyme. As we will see in
the next chapter, TGT can only perform its function as
adimer. Ligands can, therefore, interfere with the quaternary structure of the protein via their side chains. This
serious effect is either induced by ligands or it is absent
and, surprisingly, it is induced by ligands that differ by
only one methylene group in their terminal ring.
However, for the design of inhibitors for the active
pocket, it was important to see that the addition of substituents in both the 2- and 4-positions led to single-digit
nanomolar inhibitors. As afurther approach, Luzi Barandun and Florian Immekus attempted to add substituents at both positions and characterize them structurally
between Asp 102 and Asp 280 can be actively incorporated and does
not lead to acollapse of the binding afnity by disrupting the water
network (right). 21.24 is clearly seen in the differential electron density.
It forms H-bonds to Asp 280 and lls the small hydrophobic pocket
(blue arrow). Combining the results of the 2- and 4-position substi-
tutions in aderivative such as 21.25 yields subnanomolar inhibitors
(center). Again, the 2-side chains showed increased mobility in the
uracil33 pocket
(. Fig.21.17). An additional increase in potency was
observed with compounds such as 21.25 in the subnanomolar range. Thus, the inhibitory effect was below the
limit of reliable detection by the enzyme assay. From an
afnity optimization point of view, the initial goal was
achieved. However, the compounds proved to be very
large and polar. Therefore, they did not have the desired
properties for sufcient bioavailability. On the one hand,
ligands with sugar moieties in the side chain attached
in the 4-position were used for further design (cf. 21.27,
21.28, . Fig.21.20). On the other hand, aprodrug strat-
egy was pursued with the smaller ligands (not described
here).
21.10 Surprise: The Enzyme is Only
Functional as aDimer
Previous inhibitor design focused solely on blocking the
catalytic center to eliminate the function of the enzyme.
This ignored the fact that the protein must interact with
the tRNA. In the determined crystal structures, there
is always apair of two protein molecules aligned with
each other due to atwofold rotation axis in crystal packing (. Fig.21.18, yellow box). However, this does not
necessarily indicate that TGT has to exist as adimer in
. • Surprise: The Enzyme is Only Functional as aDimer
. Fig. 21.18 The enzyme TGT forms a2:1 complex with tRNA (top
right). One monomer (dark blue,1) catalyzes the base exchange in the
tRNA. The second monomer (dark green,2) holds the substrate in po-
sition for the reaction. The homodimeric arrangement has twofold C2
symmetry and is located on arotational axis in the crystal structure (yel-
low box). Acluster of four aromatic amino acids (red box, Trp 326, Tyr
330, His 333, and Phe92′ of the neighboring monomer2) is crucial for
the stability of the dimer. For reasons of symmetry, this cluster occurs
twice, as do all the other patterns. Asalt bridge of Glu 339⋯Lys52′
spans the contact surface (light blue box). Amotif consisting of aloop
solution in order to function. This picture changed when
astructure of the enzyme with a20-base RNA oligonucleotide became available. This nucleotide contains the
anticodon loop where the base exchange takes place. In
this crystal structure, the enzyme binds only one RNA
molecule as a dimer, although each monomer unit
contributes acomplete catalytic center. These centers
are both located on the same side of the homodimer.
Therefore, for steric reasons, simultaneous binding of
two tRNA substrates would be impossible. Obviously,
one monomer unit performs catalysis, while the second
monomer is responsible for the correct positioning of the
tRNA substrate for the enzyme reaction (. Fig.21.18,
upper right inset).
Next, we were interested in what determines the stability of the contact interface between the two monomer
units. Each monomer consists of 385 amino acids. The
question of stability can be investigated by targeted exchange (mutagenesis) of individual amino acids in the
contact interface. This can lead to local destabilization
of the protein contact. In this approach, we pursued the
working hypothesis that the function of this enzyme can
also be disrupted if the stability and, thus, the formation of the dimer structure is blocked. This would be an
(violet) and ahelix (light green), which occurs repeatedly with different
geometries in the numerous crystal structures with different ligands
(see . Fig.21.20, left), proves to be important for the formation of the
dimer contact (green box). (7 https://sn.pub/oZX57w)
alternative strategy to inhibit the function of TGT. But
which residues are crucial and how do they contribute to
stabilization? The contact area of both monomers spans
more than 1600 Å2 and is formed by 43amino acids with
10hydrogen bonds each, 14salt bridges, and 188 individual van der Waals contacts. Computer simulations can
help at this point. Of course, it is much easier to exchange
amino acids in the computer than experimentally by targeted mutagenesis. Stephan Jakobi rst carried out such
acomputer screening to nd the “hot spots” of contact
surface adhesion. To do this, amino acids are replaced one
after the other and MD simulations are run repeatedly.
The contribution of each amino acid to the stability of
the contact surface in the mutated variants is then analyzed. In this way, Stephan Jakobi discovered acluster
of four aromatic amino acids that turned out to be important for the stability of the dimer (. Fig.21.18, red
box). In addition, asalt bridge from Glu 339 to Lys52′
spans the contact surface (. Fig.21.18, light blue box).
To differentiate between the two monomers, adash (′)
is always added as label to the second monomer in the
following. In addition, the loop–helix motif mentioned
above (. Fig.21.18, green box), which was repeatedly
found in the crystal structures with deviating geometry,
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
21
. Fig. 21.19 Results of ahot spot analysis of the contact area cover-
ing more than 1600 Å2. The stability contributions are summarized by
abluish color coding (top right, red/green, below unfolded in top view
with color coding). The more intense and darker the blue, the stronger
the contribution of asingle amino acid to the contact (bottom right).
Acluster of four aromatic amino acids (Trp 326, Tyr 330, His 333,
and Phe92′ (yellow) on the adjacent dimer) stands out. This cluster is
shown in detail on the left. Note that the three residues Trp 326, Tyr
stood out. This led to the assumption that this motif
plays an important role in the formation of the dimer
contact (. Fig.21.19).
But how can we experimentally determine whether
and to what extent the dimer dissociates in solution? In
collaboration with Sarah Cianférani’s group in Strasbourg, France, we used native nanoESI mass spectrom-
etry. In this method, the protein is transferred from an
equilibrium solution into the gas phase and its mass is
determined from the intact protein molecule. If two or
more species are present, they can be identied by their
different masses. Since the transfer to the gas phase is
extremely fast, it is assumed that acorrect representation
of the mass ratios in solution is obtained.
330, and His 333 each form ahydrogen bond (green line) across the
interface contact surface to amino acids on the loop–helix motif of the
neighboring monomer. (7 https://sn.pub/og37OX)
21.11 Site-directed Mutagenesis:
What Binds the Dimer Together
The rst step was to successively exchange individual residues of the aromatic cluster: Trp 326 for glutamate, Tyr
330 for cysteine, His 333 for aspartate, and Phe92′ on the
other monomer also for cysteine. The choice of these residues was guided, on the one hand, by our calculations. We
also wanted to introduce more polar residues to improve
the solubility of the protein in the monomeric state. In the
wild type, the contact surface is largely hydrophobic, so
we expected adecrease in solubility when this surface is
exposed in the monomeric state. Finally, when selecting
the cysteines, we had in mind the idea of introducing new
attachment points for subsequent chemical modication
. • Site-directed Mutagenesis: What Binds the Dimer Together
. Fig. 21.20 Left Since the catalytic center and the exible loop–he-
lix motif (bottom left: orange, shown in different conformations) are
close to the dimer contact surface, inhibitors of the catalytic center
could be designed with long, spike-shaped substituents such as 21.26
that protrude into the region of the aromatic cluster and the Glu
339⋯Lys52′ salt bridge. They disrupt the geometry in this region.
Their binding increases the dissociation of the dimer into monomers.
Right With ligands 21.14, 21.27, and 21.28, we had the surprising
result that two differently packed dimers crystallized side by side. In
one, we found the usual functional dimer. In the second, the monomer
to the dimer contact surface near the aro matic cluster. We
planned to “tether” small fragments to the thiol groups
of the cysteines via achemical reaction (see Fragment
Tethering Approach, Sect.7.10). During the course of the
project, the cysteines introduced in this way proved to be
extremely instructive, as they opened the way to amodied strategy in the search for small ligands (Sect.21.15).
In afurther step, we extended our mutagenesis to the salt
bridge residues Glu 339⋯Lys52′ and Trp95. For this
purpose, double mutants have also been created.
While the wild type featured hardly any monomer in
solution, the monomer fraction increased in all variants.
This even led to mutant variants that were largely monomeric in solution. In the crystal structures of these
variants, however, we still found the C2 symmetric homodimer with only some rearrangements in the region of
units were packed together in an altered form, which formally corresponded to arotation of one monomer unit with respect to the other
one by about 130°. While the functional dimer is able to bind tRNA,
the twisted dimer is unable to do so for steric reasons (bottom right).
(7 https://sn.pub/b30k5W)
the exchanged amino acids being observed. In addition,
afew water molecules had crept into the structure in all
cases. They probably indicate how the destabilization of
the contact is initiated in aqueous solution. The fact that
we found the homodimer again and again in the crystals
can be understood as aconsequence of chemical equilibrium. As the local concentration of protein in solution
increases, as is the case at the surface of agrowing crystal,
equilibrium automatically shifts towards the dimer (Le
Chatelier’s principle). Therefore, only the dimer could be
obtained from the crystallization solution. When we performed mass spectrometric experiments with solutions
of increasing protein concentration, we also found an
increase in the dimer fraction in solution. This underlines
the concentration dependence of the dissociation in the
monomer/dimer equilibrium.
Chapter • A Case Study: Structure-Based Inhibitor Design for tRNA-Guanine Transglycosylase
21
. Fig. 21.21 Left The functionally active homodimer of TGT is
shown in the upper left. It is able to recognize and catalytically convert atRNA molecule. Right The new twisted homodimer packing
induced by ligand binding is shown on the right. It locks the enzyme
into ageometry that can no longer bind atRNA molecule. The two
dimers formally differ by arotation of about 130° of the two structurally nearly unchanged monomer units with respect to each other. The
new contact area in the twisted dimer differs signicantly from that in
the catalytically active dimer, although both are similar in size (inÅ2)
In the context of these stability studies of the contact surface interface, we were also interested in how
fast the homodimer exchanges its monomer subunits with
each other. For this purpose, we mixed aform of TGT
still bearing the so-called Strep-tag®II from apurication step at the N-terminus with an equimolar amount
of TGT without this tag. Using mass spectrometry, we
then observed the appearance of a“heterodimeric” TGT
that differed in mass and consisted of both atagged and
an untagged subunit. In the end, it was present at 50%
alongside the two “pure” TGT forms (25% each). While
virtually no “heterodimeric” TGT was found approximately two minutes after mixing the two “pure” forms,
complete exchange equilibrium was not reached until
more than 10 h had elapsed. Thus, the exchange process
is very slow. In contrast, adestabilized variant, in which
His 333 was exchanged for alanine, reached equilibrium
much more rapidly, i.e., in less than ten minutes.
and compositional inventory. However, the contact area in the catalytically active dimer is contiguous. In the twisted dimer, the interface
is divided into four separate sections (compare red surface fractions).
An important component of the interface in the catalytically active
dimer is the completion of the aromatic cluster of Trp 326, Tyr 330,
and His 333 by the addition of Phe92′ from the other dimer partner
(bottom left). In the twisted state, the role of Phe92′ is taken over by
Tyr72′ from the dimer partner, resulting in acomparable aromatic
cluster (bottom right)
21.12 When Nothing Else Works:
Chemical Poking at the Contact
Interface
Obviously, all the mutagenesis did not help to obtain
acrystal structure of the monomer. Although it became
clear that the equilibrium and the kinetics of its establishment could be shifted towards monomerization by
destabilizing the interface contact surface, what effect
did this have on the structure? Astructural feature of
TGT is that the catalytically important preQ1 binding
site is spatially very close to the dimer interface contact.
In collaboration with the group of François Diederich
in Zurich, we succeeded in synthesizing inhibitors of
the catalytic center with long needle-shaped substituents
(e.g. 21.26). This allows these ligands to spike into the
nearby dimer interface (. Fig.21.20, left). As aresult,
these spiking ligands were able to achieve at least partial
disruption of the TGT dimer. Mass spectrometry showed
an increase to approximately 25% of the dissociated species. Optimization of these spiking ligands could lead to
an alternative concept for drug development.