Protein surface recognition by dendritic ruthenium(II) tris(bipyridine) complexes

Article abstract of DOI:10.1039/b914770e

We report protein surface binding of dendritic ruthenium(II) tris(bipyiridine) complexes to α-chymotrypsin, resulting in a 1:1 and 1:2 protein complex formation as well as inhibition of the enzyme activity. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b914770e

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Protein surface recognition by dendritic ruthenium(II) tris(bipyridine)
complexesw
Junko Ohkanda,*a Ritsuko Satohb and Nobuo Katoa
Received (in Cambridge, UK) 22nd July 2009, Accepted 8th October 2009
First published as an Advance Article on the web 16th October 2009
DOI: 10.1039/b914770e
We report protein surface binding of dendritic ruthenium(II)
tris(bipyiridine) complexes to a-chymotrypsin, resulting in a
1 : 1 and 1 : 2 protein complex formation as well as inhibition
of the enzyme activity.
positioning of the functional groups and the complementary
charge distribution also have a considerable effect on its
affinity for and inhibition of ChT.
The serine protease bovine pancreatic a-ChT (pI B 9)
possesses a number of basic amino acid residues and forms a
characteristic ring structure around the active site (Fig. 1a).
The positively charged surface has been implicated in
Protein surface-directed agents have been widely investigated
due to their potential for application in pharmacology1 and
biosensing.2 Recent work in proteomimetics3 has included the
development of robust methodologies for targeting of large
protein surfaces by synthetic macromolecules,3a–c miniature
proteins,3d–f and nucleic acid-based scaffolds such as
quadruplexes3g and aptamers.3h However, the synthetic
introduction of further structural diversity into large
molecules remains a challenging problem.4 Stable metal
complexes may be of advantage in the construction of large
complexes with various functional groups from relatively
small ligand molecules.
protein–protein
interactions
with
naturally-occurring
proteinaceous inhibitors,9 and has been the target in the
development of several synthetic agents,3c dendrimers,10a and
nanoparticles.10b Based on the structural characteristics of the
surface of ChT, we introduced, at the 4 and 40 positions of
2,20-bipyridine, glutamic acid, glutamyl phenylalanine
dipeptide, and two phenylalanines with 5-amino isophthalic
acid as a spacer (1–3a; Scheme 1: see ESI for details).w The net
charge in 1–3a was fixed at ꢀ12, while the complex sizes
ranged from around 23 to 34 A in diameter. A superimposition
model suggested that 3a was of an appropriate size to cover
the active site (B600 A2) while placing the terminal carboxyl
groups near the appropriate positively charged residues on the
protein surface (Fig. 1b). A partial molecular dynamics
simulation showed that the amide bonding backbones of
1–3a were relatively rigid, as seen in the example of 1
(Fig. 1c). However, the root mean square deviation (RMSD)
for 2 was slightly larger than that of 3a (1.01, 3.39 and 2.78
for 1, 2, and 3a), indicating flexibility around the amino acid
side chains in 2. The results also suggest that a restricted
Protein self-assembly has emerged as a critical goal in the
area of nano-biomaterials. Although highly ordered protein
aggregation has been achieved,5 there are limited examples of
metal-mediated nanostructure design with protein building
blocks.6,7 Transition metal complexes with bipyridine and
similar ligands are stable under physiological conditions and
constitute a suitable scaffold for nanoarchitecture. In recent
examples, such metal ligands covalently linked to a protein
substrate and an oligopeptide have been successfully applied in
streptavidin bundles6a and peptide fibers.7 Antibody-like
metal complexes featuring large surface areas and multivalency
for protein surface recognition are expected to be useful in
protein self-assembly using a variety of proteins.
Herein we report a strategy of protein surface binding using
a series of dendritic ruthenium(II) tris(bipyridine) complexes
([Ru(bpy)3]) 1–3 (Scheme 1) in which crucial functional groups
are differently positioned for recognition of the characteristic
surface structure of a-chymotrypsin (ChT). Previous examples
of tris(bipyridine) metal complexes designed to bind to lectin8a
and cytochrome c8b have demonstrated that accumulation of
carbohydrates and carboxylates upon complexation increases
affinity with the targeted protein to a remarkable extent.
In this study, we verified that the size of the complex, the
a The Institute of Scientific and Industrial Research (ISIR),
Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
E-mail: johkanda@sanken.osaka-u.ac.jp; Fax: +81 6 6879 8474;
Tel: +81 6 6979 8472
b Department of Chemistry, Tokyo Gakugei University,
Nukuikitamachi, Koganei, Tokyo 184-8501, Japan
w Electronic supplementary information (ESI) available: Synthesis,
luminescence titration, and enzyme inhibition assay. See DOI:
10.1039/b914770e
Scheme 1 Structure of [Ru(bpy)3] complexes 1–3 and ligand 4.
Chem. Commun., 2009, 6949–6951 | 6949
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Fig. 1 (a) A view of the surface of ChT. The basic residues are
distributed around the active site. Negatively charged and positively
charged surfaces are shown in red and blue, respectively. (b) A
superimposed model of 3a (pink, CPK) and ChT. DS Viewer 6.0.
(c) Molecular dynamics simulation (10 picoseconds, 300 K) for
selected compounds (MacroModel). For the calculation, the central
ruthenium ion was replaced by a ferric ion due to the limitation of
parameters, and the six coordination bonds were omitted. The central
metal and the bipyridine rings were fixed at the initial position, except
for C3, C30, C4, and C40.
Fig. 2 (a) Titration curves for 10 mM of compounds 1 (D), 2 (J), and
3a (K) in phosphate buffer (5 mM, pH 7.4). The data set for 3a was
fitted to 1 : 1 (dashed line) and 1 : 1 and 1 : 2 equilibrium (solid line)
binding modes with ChT. Inset: Luminescence change of 3a upon
addition of ChT (ex. 470 nm). (b) A plausible model for 1 : 2 binding of
3a (CPK) to ChT (stick).
allow the formation of a rod-shaped (length 90 A) complex of
[Ru(bpy)3] with two equivalents of ChT.
Next, we evaluated the ChT inhibition activity of the
[Ru(bpy)3] complexes. The assay was performed by treating
the enzyme (1.5 mM) with 1–3 (75 mM) in phosphate
buffer (pH 7.4) followed by addition of a chromogenic sub-
strate, N-benzoyltyrosine-p-nitroanilide (BTNA, 100 mM); the
enzyme reaction was then monitored based on the change in
absorbance at 405 nm.w The proteinaceous soybean trypsin
inhibitor (STI, 20 kDa) is known to bind to ChT, which
implies an interfacial surface of 1600 A2 with a Kd value of
1 ꢂ 10ꢀ7 M.9 Only an equimolar amount of STI caused
complete inhibition of enzyme activity. Under the same
conditions, compounds 1 and 2, which were shown by titration
to bind weakly with ChT, were completely inactive and
showed moderate inhibition, respectively (inhibition percentage
at 75 mM: 0.14 ꢃ 9% and 21 ꢃ 12% for 1 and 2, respectively,
Fig. 3). In contrast, dendritic 3a showed significant inhibitory
activity (74 ꢃ 17%), which probably reflects its higher affinity
for ChT than 1 and 2. The replacement of phenylalanine
residues in 3a by lysine methyl esters (3b) or using the
corresponding bipyridine ligand (4) resulted in a remarkable
diminution of activity. The amino groups in 3b should be
protonated under the conditions (pH 7.4) due to the basicity of
the lysine e-amino group (pKa B 10.5).13 Therefore, the
electrostatic repulsion with the ChT surface is most likely
the cause of the inactivity of 3b. All of the results support the
contention that 3a recognizes the protein surface through
complementary interaction between carboxyl groups and the
positively charged amino acid residues.
conformation around the bipyridine-isophthalic spacer portions
in 3a creates a rigid binding cavity for ChT.
The binding affinity of each complex (10 mM) to ChT was
evaluated by titration, monitoring the change in luminescence
at 620 nm at pH 7.4 (Fig. 2a). Little change was observed for
1 and 2, but the relative luminescence strength of 3a increased
upon titration with ChT (Fig. 2a, inset). A simple 1 : 1 binding
model was apparently out of line with the data (dashed line),
whereas a multiple-equilibrium model with 1 : 1 and 1 : 2
binding fit well the data set (solid line), clearly demonstrating
that the association of 3a with ChT involves 1 : 1 and 1 : 2
(3a : ChT) complex formation.11 The dissociation constants for
each equilibrium step were calculated as 1.3 ꢂ 10ꢀ7 M and
4.3 ꢂ 10ꢀ7 M, respectively12 (see also Fig. S1 in ESI).w These
results suggest that the appropriately sized complex 3a binds
to the basic region of the ChT surface resulting in the
submicromolar affinity. The rigid isophthalic frameworks in
3a may also contribute to binding through an aromatic
interaction with Phe39, Phe41, and Tyr146 near the active
site. Despite their having the same net charge as 3a, the weak
affinity shown by 1 and 2 is presumably due to their smaller
sizes, especially in the case of 1, as well as the flexibility
predicted in the simulation for 2, which is of a similar size
to 3a. A plausible model for the 1 : 2 complex is shown in
Fig. 2b. Because of its spherical shape, 3a most likely possesses
two binding sites composed of three isophthalic arms at each
position. The two binding sites are in opposing positions,
separated by 1801; therefore, the first 1 : 1 binding interaction
does not affect the second interaction with ChT. Based on this,
we found it reasonable to assume that this 1 : 2 binding would
In order to examine the inhibition mode of the Ru(bpy)3
against ChT, the Lineweaver–Burk analysis was performed
for 3a.w The data set fit the noncompetitive model (Fig. 4;
Ki = 22.1 mM), showing that association of 3a with ChT does
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asymmetric complexes would be useful for obtaining structural
information about protein surfaces that is necessary for
inhibitor design. Changing the central metal ion from the
ruthenium(II) to a ferric ion would lead to a fast ligand-
exchange system, which may be applicable to a dynamic
combinatorial approach. Work towards this goal is currently
in progress.
This work was supported by the Takeda Science
Foundation, the Naito Foundation, and the Suzuken Memorial
Foundation. We thank T. Enomoto for synthesizing
compound 2, and Prof. K. Yamaguchi for mass analysis of
1 and 2. J.O. sincerely thanks Profs. H. Ishida and A. D.
Hamilton for helpful discussions and suggestion for mass
analysis of 3.
Fig. 3 Inhibition activity of compounds 1–4 (75 mM) against a-ChT
(1.5 mM). Assays were carried out using the chromogenic substrate
BTNA (100 mM) in 5 mM phosphate buffer (pH 7.4, 30 1C).
The standard deviation is given for n = 3–7.
Notes and references
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not interfere with the substrate binding to the active site.
A computationally generated model suggests that a ternary
complex formation of 3a : ChT : BTNA is possible; when 3a
binds to the targeted positively charged surface of ChT, there
still seems to be enough space available for BTNA to bind to
the active site (see Fig. S3 in ESI).w Based on the model, a
plausible explanation for the inhibition mechanism is that,
while BTNA still binds to the active site, binding of 3a to the
surface of ChT may induce the conformational change of the
protein to diminish the enzyme activity. This may account for
the relatively low inhibition activity of 3a despite of its
submicromolar affinity for ChT.
In conclusion, the dendritic ruthenium tris(bipyridine)
complex 3a presented in this paper was shown to be a strong
binder to ChT with submicromolar affinity. The observation
of 1 : 2 complex formation between 3a and ChT may lead
to further applications in protein assembly engineering.5
Compound 3a also showed inhibition activity against ChT
while the activity remains moderate. This structurally tuneable
scaffold may prove useful in the investigation of structure–
activity relationships for protein surface-directed inhibitors.
Asymmetrical structural diversity may be readily introduced
to these metal complexes using methods such as stepwise
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Fig. 4 Kinetic analysis of the inhibition of ChT by 3a. ChT was
treated with varying concentrations of 3a (0 (K), 10 (J), and
30 (.) mM) with the substrate (BTNA) concentration was increased
from 12.5 to 75 mM. T = 297 K.
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