Protein-ligand interactions: Probing the energetics of a putative cation-π interaction

Article abstract of DOI:10.1016/j.bmcl.2014.04.114

In order to probe the energetics associated with a putative cation-π interaction, thermodynamic parameters are determined for complex formation between the Grb2 SH2 domain and tripeptide derivatives of RCO-pTyr-Ac6c-Asn wherein the R group is varied to in

Full text of DOI:10.1016/j.bmcl.2014.04.114

Accepted Manuscript
Protein-Ligand Interactions: Probing the Energetics of a Putative Cation-π In-
teraction
James M. Myslinski, John H. Clements, Stephen F. Martin
PII:
S0960-894X(14)00481-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.114
BMCL 21607
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Bioorganic & Medicinal Chemistry Letters
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25 April 2014
28 April 2014
Please cite this article as: Myslinski, J.M., Clements, J.H., Martin, S.F., Protein-Ligand Interactions: Probing the
Energetics of a Putative Cation-π Interaction, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://
dx.doi.org/10.1016/j.bmcl.2014.04.114
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(HO)2OPO
CONH2
CONH2
O
O
H
N
R
X
N
N
H
H
O
Graphical Abstract

Protein-Ligand Interactions: Probing the Energetics of a Putative
Cation-π Interaction
James M. Myslinski, John H. Clements, and Stephen F. Martin*
The Department of Chemistry, the Institute of Cellular and Molecular Biology, and the Texas Institute of Drug and Diagnostic
Development, The University of Texas, Austin, Texas 78712, USA
Corresponding author e-mail: sfmartin@mail.utexas.edu
In ongoing studies directed toward correlating structure and
energetics in protein-ligand interactions, we recently became
ABSTRACT: In order to probe the energetics associated with
interested in explicitly elucidating the energetics associated with
cation-π interactions.13–18 Such interactions are important
a putative cation-π interaction, thermodynamic parameters are
determined for complex formation between the Grb2 SH2
structural features in protein folding and protein-ligand
domain and tripeptide derivatives of RCO–pTyr–Ac6c–Asn
interactions and involve a non-covalent interaction between the
wherein the R group is varied to include different alkyl,
monopole of a cationic amino group on the side chain of a Lys or
cycloalkyl, and aryl groups. Although an indole ring is reputed to
Arg residue, and the negatively charged portion of the quadrupole
have the strongest interaction with a guanidinium ion ion, binding
of the aryl group of a Tyr or Trp residue. Although the
involvement of such interactions in model systems has been
widely studied, there are relatively few investigations directed
toward quantifying the detailed energetic contributions of cation-
π interactions in protein-ligand associations. For example,
Diederich has shown that cation-π interactions contribute about
free energies, ∆G°, for derivatives of RCO–pTyr–Ac6c–Asn
bearing cyclohexyl and phenyl groups were slightly more
favorable than their indolyl analog. Crystallographic analysis of
two complexes reveals that test ligands bind in similar poses with
the notable exception of the relative orientation and proximity of
2.8 kcal mol–1 to binding free energy for complexation of ligands
the phenyl and indolyl rings relative to an arginine residue of the
domain. These spatial orientations are consistent with those
to the aromatic box of factor Xa, but binding enthalpies and
entropies were not reported.16a,b On the other hand, Marshall and
observed in other cation-π interactions, but there is no net
energetic benefit to such an interaction in this biological system.
Accordingly, although cation-π interactions are well documented
as important noncovalent forces in molecular recognition, the
energetics of such interactions may be mitigated by other
nonbonded interactions and solvation effects in protein-ligand
associations.
coworkers have found that such interactions can be mitigated by
competing, adjacent salt-bridges.18
We previously identified the SH2 domain of the growth
receptor binding protein 2 (Grb2), a cytosolic adapter protein that
participates in the Ras signal transduction pathway,19 as an
excellent model system for studying molecular recognition in a
biological system.8,11c In the context of cation-π interactions,
One of the most difficult problems in contemporary molecular
recognition involving protein-ligand interactions is understanding
how and why changes in the structures of small molecules affect
relative thermodynamic binding parameters.1 From a historical
perspective, experimental and computational studies for
associations of proteins and small molecules typically reported
Kis, IC50s, and binding free energies, ∆G, but since the advent of
isothermal titration calorimetry (ITC), binding enthalpies, ∆H,
and entropies, ∆S, are more commonly determined.2,3 As these
data have become available, paradigms to increase ligand
potency by modifying ligand structure to enhance binding
enthalpies and/or entropies are beginning to emerge.4 However,
applications of such strategies do not necessarily result in
increased affinities because of enthalpy/entropy compensation,
which may be virtually balancing,5 and because there is often no
correlation between ∆G° and either ∆H° or –T∆S°. Moreover, it
is becoming increasingly apparent that some common strategies
used to enhance protein binding entropies of small molecules are
not uniformly reliable. For example, we discovered that ligand
preorganization does not necessarily lead to enhanced protein
binding entropies, even when flexible and constrained ligands
bind in similar conformations.7–9 Although the hydrophobic effect
is generally viewed as having a favorable impact on binding
entropy,10 we and others have found that increasing the nonpolar
surface area of a ligand can eventuate in more favorable protein
binding enthalpies and less favorable binding entropies.3,11
Determining the origin(s) of such enthalpy driven hydrophobic
interactions is the subject of a number of theoretical studies.12
Furet and coworkers discovered that the affinity of 3 (IC50 = 65
nM) for the Grb2 SH2 domain was about two orders of
magnitude greater than for the related tripeptides 1 and 2, which
were approximately equipotent.20 Based upon modeling studies,
they attributed the enhanced potency to favorable stacking, or a
cation-π interaction, between the electron-rich aniline ring at the
N-terminus of 3 and the Arg67 residue of the Grb2 SH2 domain.
In a subsequent study, Nioche and coworkers found that the
related phosphotyrosine derivatives 4–6 bound with
approximately equal affinity (IC50 of 6 = 13 nM) to the Grb2 SH2
domain.21 An X-ray study revealed that the aromatic ring of the
m-amino-Cbz group of 6 in its complex with the domain aligned
in a parallel, but not completely stacked, orientation relative to
the plane of the Arg67 residue.
(HO)2OPO
(HO)2OPO
O
H
N
R
Ile–Asn–NH2
R
N
N
Asn–NH2
H
H
O
O Me
1: R = Ac
2: R = Cbz
3: R = m-H2N–Cbz
OPO(OH)2
4: R = Ac
5: R = Cbz
6: R = m-H2N–Cbz

Table 1. Thermodynamic data for complexes of 7–12 and the Grb2 SH2 domain.[a,b]
Ka
∆G°
∆H°
–T∆S°
Ligand
R
(x 106 M-1)
7.0 ± 1.2
(kcal mol-1)
–9.3 ± 0.1
(kcal mol-1)
–8.5 ± 0.4
(kcal mol-1)
–0.8 ± 0.4
Me
7
8
6.5 ± 0.1
8.8 ± 0.9
–9.3 ± 0.1
–9.5 ± 0.1
–9.3 ± 0.3
–9.1 ± 0.5
0.0 ± 0.1
O
–0.4 ± 0.3
9
O
H2N
11.4 ± 0.3
–9.6 ± 0.1
–10.0 ± 0.3
+0.4 ± 0.1
10
N
Me
23.4 ± 0.9
28.8 ± 1.3
–10.1 ± 0.1
–10.2 ± 0.1
–9.5 ± 0.5
–0.6 ± 0.1
–0.2 ± 0.1
11
12
–10.0 ± 0.5
[a]ITC experiments were conducted at 25 °C in HEPES (50 mM) with NaCl (150 mM) at pH 7.45 ± 0.05 as previously
reported.8b [b]Three or more experiments were performed for each ligand, and the averages are reported following
normalization of the n values for each experiment by adjusting ligand concentration (See Supporting Information). Errors in
the thermodynamic values were determined by the method of Krishnamurthy.2a
not appear to contribute significantly to relative binding
energetics.
In order to probe the role and detailed energetics associated
with the putative cation-π interactions between the Grb2 SH2
domain and tripeptide ligands related to
3 and 6, the
Although we sought to analyze the structures of the complexes
of 8–12 with the Grb2 SH2 domain, suitable crystals were only
obtained for complexes of 8 and 10. These structures were solved
at resolutions of 1.6 and 1.8 Å, respectively, by molecular
replacement using the structure of the domain in complex with the
parent molecule 7 (PDB code: 3S8O).23 Alignment of the
backbone atoms belonging to the domain in the complexes of 8
and 10 yields root mean square deviations (RMSDs) for all
backbone atoms of <0.2 Å (Figure 1). Although there are obvious
differences in the spatial orientations of the pTyr–1 side-chains in
the bound forms of 8 and 10, there is little variation in the
positions of equivalent atoms of 8 and 10, which superimpose
with a RMSD < 0.2 Å, which is less than the coordinate error that
is associated with the molecular models,24 following alignment of
the domains.
phosphotyrosine analogs 7–12 were prepared, and the
thermodynamic binding parameters (Ka, ∆G°, ∆H°, ∆S°) for their
associations with the Grb2 SH2 domain were determined by
isothermal titration calorimetry (ITC) (Table 1). The selection of
the Ac6c replacement for the pTyr+1 residue was predicated upon
the requirement that we wanted a series of high affinity
compounds, and the known compound 922 (IC50 = 1 nM) was
about 65-fold more potent than 3 and comparable in potency to 4–
6. Compounds 7–9 were prepared for comparison with
compounds 1–3 and 4–6 of the prior art. The indole analog 10
was selected because the indole side-chain of Trp is the aromatic
group most commonly involved in energetically significant
cation-π interactions in proteins.14c Compound 11 would enable a
comparison with the carbamate 8 and the cyclohexane analog 12
would be a control for 11.
(HO)2OPO
CONH2
CONH2
O
O
H
N
R
N
N
H
H
O
7–12
Examination of the ITC data in Table 1 reveals that 7–9 bind to
the Grb2 SH2 domain with approximately equal affinity, an
observation consistent with the findings of Nioche21 but not
Furet.20 The chain linking the carbonyl and phenyl groups of 8
has an oxygen atom, whereas there are only carbon atoms in the
linking chain of 11. This variation in atom types may result in
subtle conformational or desolvation effects that account for the
slightly more favorable binding free energy of 11, the origin of
which appears to be entropic. Furthermore, in contrast to what we
expected based upon a putative cation-π interaction, the phenyl
and cyclohexyl derivatives 11 and 12, respectively, were about
two-fold more potent than the corresponding indolyl compound
10. Given the narrow range of ∆G° values for 10–12 and
experimental error, interpreting the importance of the minor
differences in corresponding ∆H° and –T∆S° values is
problematic. However, given the close similarities in the
thermodynamic parameters for 10–12, a cation-π interaction does
(a)
(b)
Figure 1. Complexes of the Grb2 SH2 domain with 8 and 10.
Oxygen, nitrogen, and phosphorous atoms are colored red, blue,
and orange, respectively. Carbon atoms belonging to the complex
with 8 and 10 are colored magenta and cyan, respectively. (a)
Alignment of backbone atoms belonging to the domains in the
complexes, showing the domains (ribbons) and the bound ligands
(sticks). (b) The bound ligands (sticks) only. Dashed black line
represents the internal hydrogen bond between the pTyr carbonyl
oxygen atoms and the amide nitrogen atoms of the pTyr+3 sites.
In their respective complexes, both 8 and 10 make 14 direct
and five water-mediated polar contacts to the domain.25 The
cyclohexane ring at the pTyr+1 position in each complex makes a

total of 15 van der Waals (vdW) contacts with the domain.26 The
favorable nonbonded interactions between the Grb2 SH2 domain
and the atoms common to both 8 and 10 are thus virtually
identical to one another. However, because the phenyl group of 8
is positioned closer to Arg67 of the domain than the indolyl
moiety of 10, 8 makes 23 vdW contacts with the pTyr–1 subsite,
whereas 10 makes only five such contacts. However, this large
difference in the number of vdW contacts is not reflected in any
significant differences in the thermodynamic binding parameters
(Table 1).
interactions, which range from 2.8–3.3 Å, between the pTyr–1
carbonyl oxygen atoms and the two Natoms of the Arg67 side
chains in these complexes. Thus, although cation-π interactions
are well documented as important noncovalent forces in
molecular recognition, the energetics of such interactions may be
mitigated by other nonbonded interactions and solvation effects in
protein-ligand associations.
Acknowledgment. We thank the National Institutes of Health
(GM 84965), the National Science Foundation (CHE 0750329),
the Robert A. Welch Foundation (F-652), and the Norman
Hackerman Advanced Research Program for their generous
support of this research.
Cation-π interactions involving arginine side chains in proteins
tend to adopt a so-called parallel electron donor-acceptor
geometry rather than an oblique or T-shaped geometry.14c,15
Examination of the structure of the complex of 8 with the domain
reveals that the phenyl ring in the Cbz group indeed adopts such a
parallel geometry relative to the guanidinium ion of Arg67
(Figure 2a), and the carbon atoms of the phenyl ring are 3.4–3.8 Å
removed from the central carbon atom of guanidinium group. On
the other hand, the distance between the carbon atoms in the
benzenoid ring of the indole ring and the nearest nitrogen atom of
the guanidinium group in the complex of 10 range from 3.5–4.5
Å, and the planes of the guanidinium moiety of Arg67 and the
indole ring at the pTyr–1 site of 10 are oblique (Figure 2b).
Although this is not the preferred relative orientation in protein
structures, it is not possible to confidently correlate variations of
energetics with interplane angle, because there are strong cation-π
interactions having oblique geometries.14c
Supporting Information Available. Methods and materials
for ITC and X-ray crystallographic experiments, X-ray diffraction
statistics, electron density difference maps, contact diagrams, and
plots of thermodynamic data are available free of charge via the
Internet at http://pubs.acs.org.
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Å between a carbon atom of the bound ligand and a carbon, nitrogen, or
oxygen atom in the domain.

Figure(s)

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