Stapled BH3 Peptides against MCL-1: Mechanism and Design Using Atomistic Simulations
Citation: Joseph TL, Lane DP, Verma CS (
Stapled BH3 Peptides against MCL-1: Mechanism and Design Using Atomistic Simulations
Thomas L. Joseph 0
David P. Lane 0
Chandra S. Verma 0 1
Narayanaswamy Srinivasan, Indian Institute of Science, India
0 1 Bioinformatics Institute , A
1 STAR (Agency for Science, Technology and Research), Biopolis, Singapore, 3 Department of Biological Sciences, National University of Singapore , Singapore, Singapore , 4 School of Biological Sciences, Nanyang Technological University , Singapore, Singapore
Atomistic simulations of a set of stapled alpha helical peptides derived from the BH3 helix of MCL-1 (Stewart et al. (2010) Nat Chem Biol 6: 595-601) complexed to a fragment (residues 172-320) of MCL-1 revealed that the highest affinity is achieved when the staples engage the surface of MCL-1 as has also been demonstrated for p53-MDM2 (Joseph et al. (2010) Cell Cycle 9: 4560-4568; Baek et al. (2012) J Am Chem Soc 134: 103-106). Affinity is also modulated by the ability of the staples to preorganize the peptides as helices. Molecular dynamics simulations of these stapled BH3 peptides were carried out followed by determination of the energies of interactions using MM/GBSA methods. These show that the location of the staple is a key determinant of a good binding stapled peptide from a bad binder. The good binder derives binding affinity from interactions between the hydrophobic staple and a hydrophobic patch on MCL-1. The position of the staple was varied, guiding the design of new stapled peptides with higher affinities.
Competing Interests: CSV is a member of the PLoS ONE Editorial Board. This does not alter the authors strict adherence to all of the PLoS ONE policies on
sharing data and materials.
Apoptosis is a conserved process that leads to cell death.
Dysregulation of apoptosis contributes to disorders such as
malignancies . There are two recognized pathways that lead
to apoptosis: extrinsic and intrinsic . In both, a family of
Cysteine Proteases, named Caspases act in a proteolytic cascade.
The extrinsic pathway is controlled by extracellular events 
while the intrinsic pathway begins when a cell is damaged beyond
repair. The most characterized intrinsic pathway is mitochondrial
and is controlled by the B-cell lymphoma 2 (Bcl-2) protein family
. The Bcl-2 protein family comprises suppressors (e.g., Bcl-2,
Bcell lymphoma-extra large, or Bcl-XL myeloid cell leukemia
sequence 1 or MCL-1) or promoters (e.g., Bcl2 associated X
protein or Bax, Bcl-2 homologous antagonist/killer or Bak,
BH3only proteins including Bim, Bid) of apoptosis . Various
apoptotic stimuli trigger the release of factors (eg Cytochrome c)
from the mitochondria that activate caspases. Bcl-2 related
proteins appear to modulate the release of Cytochrome c .
MCL-1 is an anti-apoptotic member of the Bcl-2 family protein
 and has been shown to be expressed in different cell types .
It promotes cell survival by inhibiting the apopototic cascade and
is also found to be over-expressed in a variety of human cancers
(B-cell lymphoma, chronic lymphocytic leukemia, chronic myeloid
leukemia, etc) . Further, tumors with high levels of
antiapoptotic members of Bcl-2 such as MCL-1 are often found to be
resistant to chemotherapy . Thus, inhibition of the function of
the anti-apoptotic members of Bcl-2 such as MCL-1 may offer a
novel avenue for designing anticancer drugs [11,12].
The MCL-1 protein is 350 amino acids long and is homologous
to BH (Bcl-2 homology) domains of the Bcl-2 family . These
domains are short motifs which mediate interactions between
Bcl2 proteins in modulating apoptosis . MCL-1 has a BH3-binding
groove (Figure 1) that is made up of portions of helices a3, a4, a5
(BH1), a8 (BH2) and a2 (BH3). In addition, there is a C-terminal
transmembrane (TM) domain that localizes MCL-1 to the outer
mitochondrial membrane  which is thought to be part of the
apoptotic cascade; MCL-1 is also thought to localize to other
intracellular membranes [14,15,16].
As part of the strategy to inhibit these anti-apoptotic proteins,
Abbott developed a small molecule (ABT-737) which targets Bcl-2
and Bcl-XL with high affinity but does not target MCL-1 [17,18].
While this molecule has entered clinical trials, there are several
small molecules [19,20,21,22], peptides , and stabilized alpha
helical peptidomimetics , that inhibit MCL-1 but are still in
the investigational phases. A novel strategy to gain high affinity
peptides has been developed by Verdine & coworkers and
demonstrated its effectiveness initially for the BH3 system
(Figure 2 A and B) . This involved stabilizing a helical peptide
with an appropriately placed hydrocarbon linker which was shown
to preorganize the peptides into helices, stabilize the peptides
against proteolytic degradation and make them cell permeable. In
addition, computational models showed that the hydrocarbon
staples can gain binding energy by interacting with hydrophobic
patches on the surface of the target [26,27]. To develop such
inhibitors of MCL-1, Walensky and group identified a set of such
peptides that inhibited MCL-1 both in vitro and in vivo [25,28].
Structural characterization of the highest affinity peptide
complexed to MCL1- showed that indeed the staple interacted with a
hydrophobic part of the surface [29,30,31]. The technique of
stapling peptides has now been shown to be effective in the p53
pathway , NOTCH pathway , BCL pathway ,
estrogen activation , cholesterol efflux , and in targeting
HIV . In addition, a successful strategy employing a double
staple provides hope that this technique can also be used to recruit
longer peptides .
As we had earlier successfully predicted using molecular
dynamics (MD) simulations that the gain in affinity of the p53
peptides against MDM2 partly originated in interactions that the
hydrocarbon staples make with hydrophobic patches on MDM2
 (later validated in a crystallographic study ), we decided to
extend our studies to the report by Walensky and colleagues,
where the position of the staple along a peptide against MCL-1
was varied . MD simulations show that the interaction surfaces
can be extremely dynamic [39,40,41] and hence help guide the
careful placement of the staple in order to maximize affinity
[26,41] during the design of new peptides.
Materials and Methods
The initial structure of MCL-1 bound to a stapled peptide was
taken from the crystal structure 3MK8, resolved at 2.3 A . The
missing residues (K194-R201) were modeled using Modeler 9.7
 and guided by their positions in the NMR structure of
MCL1 bound to a peptide (PDB code 2KBW ). The starting model
included residues 172320 of human MCL-1, and residues 523
of the BH3 peptide . The stapled regions were modeled using
the Xleap module of AMBER  and the parameters were built
using the antechamber module of AMBER [45,46]. Only the
Nand C- termini of MCL-1 were capped (with acetyl or ACE and
N-methyl or NME respectively) to keep them neutral, in accord
with the experiments . Molecular dynamics simulations were
performed with the SANDER module of the AMBER9 
package employing the all-atom ff99SB force field .
Simulations were carried out for the complexes of BH3 wild type and
eleven stapled peptides bound to MCL-1 (Table 1). Each system
was solvated with a TIP3P water box  whose sides are at a
minimum distance of 10 A to any protein atom. Particle Mesh
Ewald method (PME)  was used for treating the long range
electrostatics. All bonds involving hydrogen atoms were
constrained by SHAKE . A time step of 2fs was used. Initially, the
whole system was minimized for 4,000 steps, to remove any
unfavorable interactions. Subsequently, the systems were each
heated to 300 K for 30 ps under NPT conditions. After this, each
system was equilibrated for 100 ps and then simulated for 20 ns at
constant temperature (300 K) and pressure (1 atm) and structures
were stored every 1 ps. The free energy of binding (DGbind) of the
peptides to MCL-1 was computed using the MM-GBSA
(molecular mechanics/Generalized Born surface area) method
[51,52] using the GB module  in Amber while the non-polar
component was estimated from the solvent accessible surface area
using MOLSURF  with DGsolv,np = 0.00542*SASA +0.92
. Each energy term was averaged over frames taken every 2 ps
over the last 10 ns of each simulation. Vibrational entropy was
estimated using normal mode analysis (Nmode module of Amber)
Figure 2. Structure of the staple. (A) The structure of the stapled BH3 peptide (BH3D) taken from its complex with MCL-1 as crystallized in the
xray structure (3MK8) is shown in cartoon. The staple linking amino acid positions i and i+4 is shown in sticks and the C-a atoms are shown in spheres
for clarity, (B) The chemical structure of the i, i+4 staple used.
St-XXX-St refers to the hydrocarbon linker (as shown in Figure 2B).
 and averaged over 200 ps intervals. PyMOL  and Visual
Molecular Dynamics  (VMD) were used for visualizations.
Results and Discussion
We have carried out MD studies investigating the binding of 6
BH3 peptides to MCL-1; these peptides have been experimentally
characterized by Walensky and colleagues . The peptides
include the wild type (wt) peptide and 5 stapled peptides which are
labeled MCL-SAH-A to MCL-SAH-E respectively in Figure 2c
that appears in the work reported by Walensky et al. . Our
simulations of the interactions of these peptides with MCL-1
guided the design of an additional six stapled BH3 peptides (which
we shall refer to as BH3F-BH3K; we will further refer to the wild
type peptide as BH3wt and their 5 stapled peptides as
BH3ABH3E (Table 1)). Walensky and coworkers initially designed a
peptide (BH3A) that displayed 43 nM affinity against MCL-1.
They subsequently subjected this to an alanine scan to determine
the positions where staples could be introduced, while minimizing
perturbations to the interactions with MCL-1. This yielded a set of
4 stapled peptides with affinities ranging from 1033 nM with
BH3D displaying the highest affinity for MCL-1. The complex of
BH3D bound to MCL-1 was subsequently resolved using
crystallography . This structure revealed an interaction
between the hydrophobic staple and a hydrophobic patch on
MCL-1 that was hypothesized to be responsible for part of the
enhanced affinity (Figures 3A and B); a similar feature was
predicted for the p53 stapled peptides with high affinity against
MDM2 that were characterized by the Verdine group  by our
simulation studies . Indeed, the prediction of our simulations
found close agreement in the recently described crystal structure of
MDM2 complexed to a stapled peptide , thus lending support
to our simulation strategy.
In order to benchmark our calculations, we used the crystal
structure of MCL-1 complexed to BH3D, mutated BH3D to
generate BH3A, and then subjected BH3A to a computational
alanine scan . As expected, mutating residues that are buried
(Figure S1 A and B) including L6A (L210A in the paper by
Walensky & colleagues ; henceforth the number in
parentheses will refer to this), L9A (L213A) and V16A (V220A) destabilize
the binding energies by ,35 kcal/mol. In contrast, the mutations
R10A (R214A) and D14A (D218A) undergo much greater
destabilization (,1220 kcal/mol) resulting from the loss of
extensive hbond networks that they are part of (as can be seen
in Figure S1A and Figure 3A). The computed affinities of the Ala
mutants for MCL-1 show a trend that mirrors the experimental
findings (Table S1) and establish an appropriate benchmark.
All the simulations were judged to be stable based on the time
evolution of the root-mean-square deviation (RMSD) and is given
as Figures S2, S3 and S4 and the radius of gyration of the protein
and peptides, given as Figures S5, S6 and S7. The binding
energetics show that the computed affinities of all except the
poorest peptide are similar (Table 2 and Table S2). Our
computations, in agreement with the experimental data, also
reveal BH3C as the lowest affinity peptide. The inability of the
simulations to accurately reproduce the trend in the experimental
affinity is due to the small range of experimental affinities between
BH3A and BH3D, which is 10 to 43 nM . This translates into
a free energy range of 211 to ,210 kcal/mol which is too small
to be accurately captured by current computations. While efforts
are ongoing to improve the computation of absolute binding
affinities , nevertheless the current state of the technology is
reliable only in as far as a match is obtained in the trends seen in
experiments or in some computed parameter that matches the
experimental trend. The quantitative accuracy of computations
currently are limited by various factors including force field
parameters, insufficient sampling, statistical errors, convergence,
computations of entropies [61,62,63,64], while some progress has
been reported with longer simulations in terms of sampling 
nevertheless, the simulation setup is still quite limited in its ability
to mimic experimental conditions including changing pH, salt
effects etc. Further uncertainties arise from differences in
crystallographic structures, low resolutions, incompletely resolved
structures, and lack of detailed thermodynamic decompositions of
interactions including enthalpic and entropic contributions which
could be determined using Isothermal Calorimetry combined with
Surface Plasmon Resonance. Nevertheless, simulations are a
powerful tool to yield structural insights that rationalize observed
trends as has also been shown in several other systems [26,32,38]
and are proving useful to guide new experiments .
We first examine the complex of BH3D, the peptide with the
highest affinity against MCL-1. This peptide was derived from the
a2 helix (208KALETLRRVGDGVQRNHETAF228) of the BH3
domain of MCL-1. In the complexed state, it exists as a short
Figure 3. The structure of MCL-1 (shown in grey) bound to BH3D peptide taken from the crystal structure 3MK8 . (A) The Arg10
sidechain is stabilized by the His252 backbone. The interaction of Asp14 with Arg263, and the hbond cluster comprising the sidechains Asp256,
Asn260 and Arg263 with Asp14 are well maintained (shown in cartoon), (B) The packing of the BH3D staple against the hydrophobic residues of
MCL1 shown in surface.
amphipathic a-helix, engaging the BH3-binding groove of MCL-1
with additional contacts between the staple and a hydrophobic
patch on MCL-1 (Figures 3A and B). Although variants of BH3
that are active against MCL-1 have been reported [24,43,67,68],
BH3D displays the highest affinity. The crystallographic data 
shows that hydrophobic residues Leu6, Leu9, Val12 and Val16 of
BH3D lie buried deep inside the hydrophobic groove of MCL-1.
These interactions are further strengthened by several hbond
networks. These include: sidechain of Asp14 in BH3D makes
hbonds with sidechains of Arg263 and Asn260 of MCL-1, and
Arg263 in turn engages in a salt bridge with Asp256 of MCL-1;
sidechain of Arg10 of BH3D hbonds with Ser255 sidechain and
His252 backbone of MCL-1; BH3D Glu7 makes an hbond with
His252 of MCL-1 and, NE2 in His20 of BH3D hbonds with
backbone of Phe318 of MCL-1.
Our simulations reveal similar hbonding patterns along with
some differences. The Glu7-His252 interaction is replaced with
His252 making a transient hbond with the backbone of Leu6 while
the Glu7 sidechain prefers to be solvated with an occasional salt
bridge with Arg11. The Arg10 sidechain is stabilized by the
His252 backbone throughout the simulation. The interaction of
Asp14 with Arg263, and the hbond cluster comprising the
sidechains Asp256, Asn260 and Arg263 with Asp14 are stable
throughout the 20ns. The interaction of His20 sidechain with the
Phe318 backbone exists for 96% of the simulation time (Figures 3A
It is clear that the peptide is well sequestered in the binding
pocket and doesnt undergo any large conformational
rearrangements. The reproduction of the crystallographically observed
characteristics of the interactions between the peptide and the
receptor suggest that the simulations are well behaved.
Peptides in Solution
All simulations were judged to be stable based on the time
evolution of the RMSD (Figure S3) and radius of gyration (Figure
S6). Root-mean square fluctuations (RMSF) (Figure S8) of all
peptides remain similar and as expected, the regions constrained
by the staples show lower fluctuations. It is interesting that the
poor binding peptide BH3C shows higher helicity compared to the
good binders like BH3A, BH3D and BH3E peptides (Figure S9A
In the wt peptide simulations (Figure S10A), the helicity extends
from Leu6Gln17 (crystallographically observed) to Leu6Glu21.
This results from hbonds between the sidechains of Asp14 and
Arg18. The positively charged Arg18 interacts with the negatively
charged Asp14 and this is complemented by an hbond between
Thr18 and Asp21. The backbone of Arg18 forms an hbond with
the sidechain of Thr22, which makes this peptide more helical at
its C-terminus. At the N-terminus, the Thr8 sidechain interacts
with the backbone of Ala5 to make this region helical too. In
BH3A, Arg11 is replaced with the staple, which makes this peptide
more negatively charged and with somewhat reduced helicity
(compared to BH3wt). The staple also removes stabilizing
interactions of Glu7 with Arg11 and renders the region highly
mobile; with the staple localized to the middle of the helix, the
charged ends prefer to be solvated and hence are highly mobile. In
BH3B, the staple is located at the N-terminus which makes this
region more helical; the interactions of the charged residues
Asp14, Arg18 and Glu21 at the C-terminus make this region more
267.4(5.3) 266.8(6.2) 262.5(8.3) 252.9(5.2) 263.2(4.7) 268.1(4.6) 264.6(4.9) 268.2(5.4) 268.7(4.7) 271.7(5.9) 270.3(4.5) 272.7(4.7)
38.3(5.6) 38.5(4.5) 39.0(5.3)
helical; the intervening region is not helical. BH3C (Figure S10B)
peptide is the most helical among all the unbound peptides
analyzed, and interestingly is also the only inactive peptide. In this
peptide the staple is placed in the middle of the entire sequence.
The increased helicity arises from enhanced helicity at the
terminal regions promoted by stable interactions between the
sidechains of Glu7 and Arg11 and between the sidechains of
Arg18 and Glu21. The localization of the staple in the centre
prevents Asp14 from interacting with Arg11 and Arg18. In BH3D
(Figure S10C), Glu21 is replaced with the staple, which makes this
peptide positively charged and also has somewhat reduced helicity
(compared to BH3wt). The removal of Glu21 removes the
stabilizing interactions of Arg18 and this in turn interacts with
negatively charged Asp14. The staple also appears to lead to an
hbond between the sidechain of Thr22 and the backbone of
Arg18, further imparting helicity to the C-terminal region. While
in the BH3E peptide, although the staple is located in the
Cterminal region, the charged residues in the N-terminal region
appear to induce helicity in this region resulting in helicity in both
the N-terminal and C-terminal regions; the region in between
remains unstructured or in a loop conformation. In summary, all
peptides are equally helical; BH3C is most helical and yet least
Simulations of the MCL-1-Peptide Complexes
Secondary structure of the peptide in the complex. In
contrast to the peptides in solution, when complexed to MCL-1,
all peptides except BH3C are helical (Figures S11AL), especially
in the Glu7Thr22 region (throughout the 20 ns). So what is the
reason for this paradoxical behavior?
We find that upon complexation, the staple in the poorest
binder BH3C is located in a position which leads to maximal
disruption of the hbond network that has been highlighted above.
The introduction of the staple at Gly13 leads to a loss of the
hbonds that are made between Asp14 of the peptide and Asn260/
Arg263 of MCL-1; the sidechain of Asp14 interacts instead with
the Arg18 sidechain in BH3C, leading to a strain that results in
reduced helicity of BH3C in its C-terminal region. However, the
His252 backbone-Arg10 sidechain, Ser255 sidechain-Arg10
sidechain, and His20 sidechain-Phe318 backbone interactions are
conserved, albeit with a reduced lifetime. The other end of the
staple replaces Gln17 and this leads to a loss of the hbond with
Gly262. In general, all the peptides, except BH3C, show improved
helicity in the bound state relative to their free states, as is evident
from the temporal evolution of the secondary structures (Figures
S11AL); BH3B, BH3D and BH3H are most helical.
Key interactions in the MCL-1-Peptide
complexes. Overall RMSF of all the complexes remain similar
(Figures S12 and S13) the only real differences are seen in the
peptides. As expected, the peptides show lower fluctuations either
at the 3 amino acids, Leu6, Leu9 and Val26 that are deeply
embedded in MCL-1 or in the regions that are constrained by the
The WT simulation shows the 819 region as helical
throughout the 20 ns, as also is the case in BH3B, BH3D,
BH3F and BH3H. The other peptides show the following regions
as helical: 1121 (BH3A), 721 (BH3E), 719 (BH3G), 722
(BH3I), 1021 (BH3J and BH3K). This appears to be in accord
with Walensky et al. , who designed the peptides with a view to
achieving higher affinity through enhanced helicity of the peptides,
especially by the introduction of i, i+4 staple. Similar features
characterized the design and affinities of peptides for the
p53MDM2 and estrogen receptor systems [26,32,38].
In the MCL-1 - BH3wt complex (Figures 4A, 4B) the
interactions that engage Arg10 and Asp14 and Leu6, Leu9 and
Val16 in BH3D (Figures 3A and B) are maintained. There are
additional interactions (legend to Figures 4A and B) and (Movies
S1 and S2). The charged residues Arg11, Arg18 and Glu21 prefer
to be solvated. Significant contributions to the binding energy are
made by key hydrophobic residues Leu6, Leu9, Val12 and Val16
(25.5, 24.9, 23.2 and 22.9 kcal/mol respectively), and by polar
residues Arg10, Asp14 and Gln17(25.4, 23.1 and 21.7
respectively), (Table 2; Tables S2 and S3).
From the simulations of the complexes, visual inspection
immediately shows that the good binders and the poor binder
can be separated based on the location of the staples as this
appears to determine their orientations and the associated
interactions in the complexes.
In the non-binder (BH3C), the staple is located in order to avoid
clashes with the surface of MCL-1, but the peptide is distorted
from helicity. In contrast, the good binders have the peptides in
helical conformations and their staples either draped over the
surface of MCL-1, or in close proximity and clearly enhance the
affinity of the peptides by these additional interactions (Table 2).
There appear to be two major drivers of the high affinities: (a) gain
in interaction energy of the peptide as a result of the MCL-1-staple
interaction; (b) decrease in the penalty paid for hydrating the
hydrophobic staple. In addition, there is the reduced entropic
penalty for immobilizing the peptides onto the surface of MCL-1
by the staple-induced pre-organization into helical motifs as we
have seen in the section on peptide simulations.
BH3 peptides [24,43,67] derived from BID, BIM and NOXA
have also been shown to bind to MCL-1, and have hydrophobic
residues at homologous positions. Residues I86/I148, L90/L152/
L78, V93/I155/I81 and M97/F159/V85 are buried deeply inside
the BH3 binding groove of MCL-1; in Walenskys BH3 peptides,
Leu6, Leu9, and Val16 are buried while Val12 is partially
In BH3wt, Arg11 and Gly15 make no contribution to the
binding since Arg11 is well solvated. Hence replacing these
residues to form the staple of BH3A would in principle be
tolerated (Figures S1A and B). Simulations show that helicity of
BH3A decreases in its N-terminal region (Figure S11B). Arg10 and
Asp14 maintain the hbond cluster as seen for BH3D. The
hydrocarbon staple is solvent exposed, but it contributes to the
binding significantly (21.9 kcal/mol); in contrast Arg11 and
Gly15 contribute negligibly in BH3wt. In BH3A, significant
contributions were made by key hydrophobic residues Leu6, Leu9,
Val12 and Val16 (25.5, 25.0, 23.0 and 23.1 (kcal/mol)
respectively), and also by polar residues Arg10, Asp14 and
Gln17 (25.3, 23.4 and 22.2 (kcal/mol) respectively), to the
binding energy. Overall the computed binding affinity of BH3A is
similar in strength to that of BH3wt (Table 2; Tables S2 and S3).
In BH3B, the staple replaces Glu7 and Arg11, which results in
better helicity. With the staple pointing into solvent, the
interactions remain similar to those of BH3A (Figures S14A and
B). The Glu7 sidechain makes transient interactions with the
Nterminal in BH3wt, which is lost upon the introduction of the
staple at position 7. This constrains the Nterminal region into a
helical state (Figure S11C), leading to reduced mobility. The
presence of the staple reduces the interactions of Arg10 with
His252 and Ser255 (the lifetimes are reduced from 89% to 75% of
the simulation time) Asp14 maintains the network seen for BH3D
whilst Gln17 makes an hbond interaction with Gly262.
The staple contributes 1 kcal/mol more than the staple in
BH3A. However Glu7 contributes ,1.6 kcal/mol in BH3A and
so the net result of replacing Glu7 by the staple in BH3B is actually
Gly262 (shown in cartoon) (H) The hydrophobic groups Leu6, Leu9, and Val16 are buried in the hydrophobic binding groove on the surface of MCL-1
(shown in surface).
destabilizing compared to BH3A. In the BH3B peptide, significant
contributions were made by key hydrophobic residues i.e., Leu6,
Leu9, Val12 and Val16 (25.7, 25.1, 22.9 and 23.2 (kcal/mol)
respectively), and also by polar residues i.e., Arg10, Asp14 and
Gln17 (24.3, 22.9 and 22.2 (kcal/mol) respectively) to the
binding energy. The overall computed free energy is similar to
WT, in agreement with binding affinities (Table 2; Tables S2 and
In BH3C, the staple replaces Gly13 and Gln17, the location of
the staple forces it to point into the MCL-1 surface creating a steric
clash and thus a strain on the backbone of the BH3C peptide
which prevents the interaction with MCL-1, unlike with the other
In the wtBH3, Gln17 makes a strong hbond with Gly262
backbone, contributing ,2 kcal/mol to the binding. This
positions the staple (in place of Gly13-Gln17) into a potential
clash between the peptide and MCL-1. This is alleviated by a
conformational rearrangement such that Asp14 pulls away from
Arg263, and forms a salt bridge with Arg18. The net result is
increased strain in the peptide, helical conformation in the stapled
region and poor helical content in the terminal regions (Figure
S11D). The loss of key hbond networks result in decreased
contributions from Arg10 (23.6) and Asp14 (20.1), when
compared with BH3wt peptide (Figures 4C and 4D; Movies S3
and S4). The overall binding energy of BH3C peptide is reduced
(,16 kcal/mol) significantly compared with BH3wt (Table 2;
Tables S2 and S3).
In BH3D, the staple bridging positions 17 and 21 results in a
better overall helicity with retention of key interactions, with a part
of the staple draped over MCL-1. In the wtBH3 sidechain of
Gln17 makes a strong hbond with the Gly262 backbone; while
sidechain of Glu21 interacts with the sidechain of Gln17.
Replacing these residues with the staple derives additional
hydrophobic contacts from neighboring residues Asn260,
Trp261, Gly262, Phe318 and Phe319. The overall mobility of
the peptide is significantly reduced in the C-terminal region
(Figures 3A and B; Movies S5 and S6).
In the BH3D peptide, significant contributions were made by
key hydrophobic residues i.e., Leu6, Leu9, Val12 and Val16
(24.7, 24.8, 23.1 and 22.8 kcal/mol respectively), and also by
polar residues i.e., Arg10 and Asp14 (25.3 and 22.8 kcal/mol
respectively) to the binding energy. The hydrocarbon staple
contributes significantly (24.3 kcal/mol), equivalent to that of
Leu6 and Leu9. The contribution of the staple is highest among all
the peptides. The contribution of Gln17 in BH3 wt is 21.7 kcal/
mol, clearly suggesting that the staple contributes an extra
,2.5 kcal/mol (Table 2; Tables S2 and S3).
The BH3E staple stabilizes the C-terminal region by reducing
its mobility. However this staple is less packed against the surface
of MCL-1 compared to the BH3D staple. The position of the
BH3E enables Gln17 to make hbond interactions with the Gly262
backbone (Figures S14C and D). Significant contributions were
made by key hydrophobic residues i.e., Leu6, Leu9, Val12 and
Val16 (25.3, 25.0, 23.0 and 23.2 kcal/mol respectively), and
also by polar residues i.e., Arg10 and Asp14 (24.5 and 23.2 kcal/
mol respectively) to the binding energy (Table 2 and Tables S2
and S3). The hydrocarbon staple contributes an excess
(21.0 kcal/mol) over residues it replaces in BH3wt.
In conclusion, we find that amongst the peptides designed by
Walensky et al. , the tightest binder BH3D retains the hbond
networks that are characteristic of the interactions of the wild type
(except for the hbond between the sidechain of Gln17 with the
backbone of Gly262) and its staple packed against the MCL-l
surface when compared with other staples reported.
Designing New Peptides with Higher Affinity
Computational alanine scanning. Guided by the above
findings, we next attempt to design peptides with higher affinities.
We first use computational alanine scanning , on BH3wt
whereby the orientation of the mutated sidechain is energetically
optimized using SCWRL [69,70], and the effects of this on the
structure and interactions of MCL-BH3wt are computed. We also
subject these calculations to 1 ns simulations each; however the
simulations did not converge, so before investing in longer
simulations, we decided to use the results arising from energy
minimizations. L6A, L9A and V16A mutations were found to
have reduced affinity for MCL-1 by 3-5 kcal/mol (Table S4),
reflecting the importance of the larger sidechains Val and Leu in
the hydrophobic interactions with the surface. In addition, R10A
and D14A also were associated with loss in binding affinity
because of the loss in their hbond networks. This motivated us to
vary the staple points across the other residues as their changes do
not seem to perturb the affinity.
New staple positions and mutations. We noticed that in
all the simulations, Arg18 interacts with both Glu21 and Thr22
transiently. So we reasoned that mutation of Thr22 to Asp may
enhance the stability by forming a salt bridge between Arg18 and
Asp22. This peptide, called BH3F (Figures S14E and F) did not
yield any improvements because the charged residues only
interacted transiently and preferred to remain solvated (Table 2;
Tables S2 and S3); some reduction in mobility at the C-terminus
We modified BH3B (the staple is across positions 7 and 11) and
added an extra staple that linked the Arg18 and Thr22 positions at
the C-terminus; double stapling has been used successfully in the
context of longer peptides . In this peptide, called BH3G, both
staples remain solvent exposed, and did not interact significantly
with the MCL surface. However, the staples stabilize the helicity
when compared with the wt type MCL-1. The hbond clusters
between the peptide and the protein were well maintained (Figures
S14G and H). In the BH3G peptide, significant contributions were
made to the overall binding energy of 226.4 kcal/mol by
hydrophobic residues i.e., Leu6, Leu9, Val12 and Val16 (25.3,
24.8, 23.2 and 22.7 (kcal/mol) respectively) and also by polar
residues i.e., Arg10 and Asp14 (25.1 and 23.2 (kcal/mol)
respectively) to the binding energy. In addition, the N-terminal
staple, being closer to the surface of MCL-1, contributed
23.1 kcal/mol whereas the C-terminal staple contribution was
negligible (20.4 kcal/mol).
In contrast to the observation that the key hydrophobic residues
Leu6, Leu9 and Val16 present in the BH3 peptides are embedded
into the hydrophobic pocket on the surface of MCL-1, Val12 is
partially exposed. Val12 is surrounded by His224, Phe228,
Met231 and Phe270 which is a hydrophobic patch and hence
offers an opportunity for exploitation by the introduction of a
staple in the vicinity of these residues. To explore this, we took
BH3D and added a second staple that linked Thr8 with Val12
(called BH3H). The binding affinity of this peptide improved by
,7 kcal/mol compared to BH3D and mostly arose from the
improved packing of this stapled region against His224, Phe228,
Met231 and Phe270. Our simulations suggest that this region
offers a well defined hydrophobic patch (Figures 4E and 4F;
Movies S7 and S8). The overall binding energy (233.4 kcal/mol)
comprises contributions from hydrophobic residues i.e., Leu6,
Leu9 and Val16 (23.8, 24.5 and 23 (kcal/mol) respectively) and
also by polar residues i.e., Arg10 and Asp14 (25.9 and 23 (kcal/
mol) respectively). The N-terminal staple that was introduced,
contributed 27.3 kcal/mol (the highest so far amongst the staples)
while the C-terminal staple (BH3D staple) contributed 24.5 kcal/
mol. Together these two staples contribute ,33% of the overall
binding energy (Table 2; Tables S2 and S3).
To further optimize BH3H, we noticed that Ala5 lies in the
vicinity of Lys234 (Figures 4E and F) and so we mutated it to Asp5
(called BH3I) to introduce a potential salt bridge. We further
mutated Arg18 to Asp (called BH3J) in order to reduce the
mobility at the C-terminus. However, the Asp5-Lys234 interaction
was only transient in both BH3I and BH3J, but the mobility of
BH3J was reduced. The associated binding energies were 233.5
and 231.7 kcal/mol respectively (Table 2; Tables S2 and S3).
Clearly these changes did not result in any significant differences in
the affinity compared to BH3H (Figures S14 I, J, K and L).
We finally took BH3H and removed the C-terminal staple (the
one introduced by Walensky in BH3D) to examine the interactions
of the peptide with only an N-terminal staple. We find that this did
not disturb the hbond cluster between the protein and the peptides
(Figures 4G and 4H; Movies S9 and S10). Indeed, the removal of
the BH3D staple brings back Gln17 which stabilizes the system by
hbonding to the backbone of Gly262, as in the wild type system.
The overall binding energy surprisingly remains 233.7 kcal/mol
and contributions from hydrophobic residues i.e., Leu6, Leu9 and
Val16 are 25.5, 24.5 and 22.9 kcal/mol respectively while those
from polar residues i.e., Arg10 and Asp14 are 25.8 and
23.2 kcal/mol respectively. The staple contributed 27.4 kcal/
mol, as in BH3H. This clearly shows that the effects of the staples
at the two termini are decoupled from each other.
The staple connecting Gln17-Glu21 in BH3D contributes
24.3 kcal/mol to the binding, while in wild type, the contribution
of Gln17 is 21.7 kcal/mol. Thus the net contribution of this staple
is ,2.5 kcal/mol. However, in BH3K (or indeed in BH3H), the
staple connecting Thr8-Val12, contributes ,7.3 kcal/mol which
is much higher (the contributions of Thr8 and Val12 are 20.4 and
23.2 kcal/mol, totaling ,3 kcal/mol less than the staple that
replaces them). Moreover this peptide also has the Gln17
sidechain making hbond with Gly262, and contributes
,2 Kcal/mol to binding energy (Table 2; Tables S2 and S3).
Inhibition of protein-protein interactions and modulation of
associated signaling is slowly gaining popularity as progress is
made in areas of fragment based drug discovery [72,73],
peptidomimetics  etc. The latest addition to this collection,
that is demonstrating great promise, are stapled peptides
[24,28,32]. So far, these have been most effective in targeting
proteins where the target site requires a helical motif in its binding
partner. Stapled peptides appear to be excellent at this since they
already are preorganzied into a helical fold, thus reducing the
entropic costs of localization [24,28,32,33].
Walensky et al. , have optimized the BH3 helix through
stapling as a potent MCL-1 inhibitor. They demonstrated through
structural studies that the staple derives additional binding energy
by interacting with a hydrophobic patch on the MCL-1 surface.
MD simulations of these peptides show that (a) the location of the
staple is a key determinant of good from bad binder. (b) the good
binders derive binding affinity from interactions between the
hydrophobic staple and hydrophobic patches on the MCL-1
surface; indeed the contribution to the binding energies due to
these interactions can be as large as that contributed by the buried
residues (c) there are peptides that bind with higher affinity but the
staples appear to point out into solvent (BH3A/BH3B); these
staples seem to push the peptide into the binding site, yielding
tighter interactions of the buried residues (Table S3) and are
similar to observations made elsewhere ; indeed, stapled
peptides against the HIV capsid protein have also been shown by
NMR to have the staples pointing into solvent ; the
observation that BH3C is most helical in solution and yet the
worst binder appears paradoxical at first glance and yet upon
scrutiny reminds and educates us that the interaction between
protein and peptide is modulated by very dynamic surfaces [41,76]
and that perhaps creation of too tight a helix in the peptide in
solution will hinder an efficient capture and binding of the peptide
by the target protein surface. Clearly more detailed studies on
diverse systems will appear in the near future and give us glimpses
into structure-activity relationships between the amino acid
compositions of peptides, the optimal locations of staples, the
ability to enter cells unaided and the mechanisms of these exciting
molecules which hold promise as a new class of reagents for
interrogating biology and as therapeutics. For now, guided by the
findings of Walensky and his group, and the insights offered by the
MD simulations, we have carried out mutagenesis to design
peptides that computationally demonstrate higher affinities for
MCL-1 and are currently being tested in the laboratories of
Figure S1 BH3A bound to MCL-1 (shown in grey). (A)
R10 sidechain makes hbonds with the H252 backbone and the
S255 sidechain, while the D14 sidechain makes hbonds with the
sidechains of N260 and R263 (shown in cartoon), (B) The
hydrophobic residues are deeply buried inside MCL-1 (shown in
Radius of gyration for the BH3 peptides in
Radius of gyration for the BH3 peptides in
Figure S9 Temporal evolution of the secondary
structure profiles of BH3 peptides over 20 ns in solution (A)
BH3wt; (B) BH3A; (C) BH3B; (D) BH3C; (E) BH3D; (F)
BH3E; (G) BH3F; (H) BH3G; (I) BH3H; (J) BH3I; (K)
BH3J and (L) BH3K. It is clear that the wild type peptide
assumes a largely helical conformation in solution, especially in the
Figure S10 Solution structures of unbound the BH3
peptides. (A) Helicity is observed in the regions of Leu6Glu21
in BH3wt. (B) BH3C peptide is the most helical among all the
unbound peptides analyzed, and interestingly is also the only
inactive peptide and (C) Helicity in the BH3D peptide extends
from Leu6Gln17 (crystallographically observed) to Leu6Glu21.
Figure S11 Temporal evolution of the secondary
structure profiles of the BH3 peptides when bound with
MCL1 over 20 ns in solution. (A) BH3wt; (B) BH3A; (C) BH3B; (D)
BH3C; (E) BH3D; (F) BH3E; (G) BH3F; (H) BH3G; (I) BH3H; (J)
BH3I; (K) BH3J and (L) BH3K. When complexed to MCL-1, all
peptides except BH3C are helical, especially in the Glu7Thr22
Figure S13 Root mean squared fluctuations for the BH3
peptides in complexes. BH3C peptide show higher
fluctuations when compared with all other peptides.
Figure S14 BH3B bound to MCL-1 (shown in grey). (A)
Asp14 maintains the hbond network with Arg263, whilst Gln17
makes an hbond interaction with Gly262. (shown in cartoon), (B)
The hydrophobic groups Leu6, Leu9, and Val16 are buried in the
hydrophobic binding groove on the surface of MCL-1 (shown in
surface); BH3E bound to MCL-1 (shown in grey) (C) BH3E staple
is less packed against the surface of MCL-1 compared to the staple
in the best binder BH3D, but the positioning of the staple enables
Gln17 to make hbond interactions with the Gly262 backbone
(shown in cartoon), (D) The hydrophobic groups Leu6, Leu9, and
Val16 are buried in the hydrophobic binding groove on the
surface of MCL-1 (shown in surface); BH3F bound to MCL-1
(shown in grey) (E) Interactions similar to those made by BH3D
are also observed in the BH3F peptide bound to MCL-1, with the
staple enabling Gln17 to make hbond interactions with the Gly262
backbone (shown in cartoon), (F) The hydrophobic groups Leu6,
Leu9, and Val16 are buried in the hydrophobic binding groove on
the surface of MCL-1 (shown in surface); BH3G bound to MCL-1
(shown in grey) (G) Double stapling improves the packing of the
stapled regions and also maintains the helical content (shown in
cartoon), (H) The hydrophobic groups Leu6, Leu9, and Val16 are
buried in the hydrophobic binding groove on the surface of
MCL1 (shown in surface); BH3I bound to MCL-1 (shown in grey) (I)
Double stapling improves the packing of those stapled regions and
also maintains the helical content (shown in cartoon), (J) The
hydrophobic groups Leu6, Leu9, and Val16 are buried in the
hydrophobic binding groove on the surface of MCL-1 (shown in
surface); BHJ bound to MCL-1 (shown in grey) (K) Double
stapling improves the packing of the stapled regions and also
maintains the helical content (shown in cartoon), (L) The
Table S1 Binding enthalpies (kcal/mol) of BH3A
stapled peptide against MCL-1 using single point
computational alanine scanning.
Table S3 Residuewise energy contributions (in kcal/
mol) of BH3 peptides for its interactions with MCL-1.
Movie S1 Movie of MD simulation trajectory of BH3wt
bound to MCL-1 (MCL-1 is shown in cartoon).
Movie S2 Movie of MD simulation trajectory of BH3wt
bound to MCL-1(MCL-1 is shown in surface).
Movie S3 Movie of MD simulation trajectory of BH3C
bound to MCL-1 (MCL-1 is shown in cartoon).
Movie S4 Movie of MD simulation trajectory of BH3C
bound to MCL-1 (MCL-1 is shown in surface).
Movie S5 Movie of MD simulation trajectory of BH3D
bound to MCL-1 (MCL-1 is shown in cartoon).
Movie S6 Movie of MD simulation trajectory of BH3D
bound to MCL-1 (MCL-1 is shown in surface).
Movie S7 Movie of MD simulation trajectory of BH3H
bound to MCL-1 (MCL-1 is shown in cartoon).
Movie S8 Movie of MD simulation trajectory of BH3H
bound to MCL-1 (MCL-1 is shown in surface).
Movie S9 Movie of MD simulation trajectory of BH3K
bound to MCL-1 (MCL-1 is shown in cartoon).
Movie S10 Movie of MD simulation trajectory of BH3K
bound to MCL-1 (MCL-1 is shown in surface).
Conceived and designed the experiments: TLJ DPL CSV. Performed the
experiments: TLJ CSV. Analyzed the data: TLJ DPL CSV. Wrote the
paper: TLJ DPL CSV.
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