Protein Surface Mimetics: Understanding How Ruthenium Tris(Bipyridines) Interact with Proteins

Article abstract of DOI:10.1002/cbic.201600552

Protein surface mimetics achieve high-affinity binding by exploiting a scaffold to project binding groups over a large area of solvent-exposed protein surface to make multiple cooperative noncovalent interactions. Such recognition is a prerequisite for competitive/orthosteric inhibition of protein–protein interactions (PPIs). This paper describes biophysical and structural studies on ruthenium(II) tris(bipyridine) surface mimetics that recognize cytochrome (cyt) c and inhibit the cyt c/cyt c peroxidase (CCP) PPI. Binding is electrostatically driven, with enhanced affinity achieved through enthalpic contributions thought to arise from the ability of the surface mimetics to make a greater number of noncovalent interactions than CCP with surface-exposed basic residues on cyt c. High-field natural abundance ¹H,¹⁵N HSQC NMR experiments are consistent with surface mimetics binding to cyt c in similar manner to CCP. This provides a framework for understanding recognition of proteins by supramolecular receptors and informing the design of ligands superior to the protein partners upon which they are inspired.

Full text of DOI:10.1002/cbic.201600552

Accepted Article
Title: Protein surface mimetics: understanding how ruthenium tris(bipyridines) inter-
act with proteins
Authors: Sarah H Hewitt; Maria H Filby; Ed Hayes; Lars T Kuhn; Arnout P. Kalverda;
Michael E Webb; Andrew John Wilson
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ChemBioChem
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Protein surface mimetics: understanding how ruthenium
tris(bipyridines) interact with proteins
Sarah H. Hewitt, Maria H. Filby, Ed Hayes, Lars T. Kuhn, Arnout P. Kalverda, Michael E. Webb and
Andrew J. Wilson*
Abstract: Protein surface mimetics achieve high affinity binding by
exploiting a scaffold to project binding groups over a large area of
solvent exposed protein surface to make multiple co-operative non-
covalent interactions. Such recognition is
a pre-requisite for
competitive/ orthosteric inhibition of protein-protein interactions
(PPIs). This paper describes biophysical and structural studies on
ruthenium(II) tris(bipyridine) surface mimetics that recognize
cytochrome (cyt) c and inhibit the cyt c/ cyt c peroxidase (CCP) PPI.
Binding is electrostatically driven, with enhanced affinity achieved
through enthalpic contributions thought to arise from the ability of the
surface mimetics to make
a greater number of non-covalent
interactions with surface exposed basic residues on cyt c in
comparison to CCP. High field natural abundance 1H-15N HSQC
NMR experiments are consistent with surface mimetics binding to
cyt c in similar manner to CCP. This provides a framework for
understanding recognition of proteins by supramolecular receptors
and informing the design of ligands superior to the protein partners
upon which they are inspired.
Introduction
Protein-protein interactions (PPIs) are considered difficult to
inhibit using conventional synthetic molecules;[1,2] they typically
involve large interfaces with few discernable pockets on either
partner that represent the hallmark of traditional ligandable[3]
proteins.[4] Given conventional approaches for ligand discovery
e.g. high-throughput screening and fragment based drug
discovery have met with limited success in identifying PPI
inhibitors,[5,6] supramolecular chemical biology[7] with a focus on
understanding and controlling molecular recognition is well
placed to elaborate new strategies. One such strategy is the
surface mimetic approach;[8–10] protein surface mimetics are a
class of molecular structure that utilize a scaffold to project
multiple binding groups over a large area of protein surface and
achieve high affinity protein binding. Several different scaffolds
have been used as protein surface mimetics, including
calixarenes,[11–15] porphyrins,[8,16–18] dendrimers,[19–23] metal
complexes,[9,24,25] nanoparticles,[26–28] and others.[29–33]
Figure 1. The Ru(II)(bpy)3 surface mimetics and their PPI counterparts, cyt c
and CCP (A) Ru(II)(bpy)3 complexes 1 and 2, (B) the cyt c/ CCP interaction,
cyt c in red, CCP in purple (PDB ID 1U75)[34] and (C) the interaction faces of
cyt c (left) and CCP (right), showing a ring (red circle) of basic amino acid
residues (blue) on cyt c and a complementary patch (blue circle) on CCP with
acidic amino acid residues (red)
We and others previously introduced highly functionalized
ruthenium(II) tris(bipyridine) (Ru(II)(bpy)3) complexes as protein
surface mimetics.[35–41] These large, multivalent, luminescent
molecules have
a chemically inert core, which can be
peripherally functionalized with different binding groups in a
stereochemically and geometrically rich manner. Hamachi and
[a]
Miss S. H. Hewitt , Dr M. H. Filby, Dr E. Hayes Dr M. E. Webb Prof
A. J. Wilson
coworkers initially designed
a
carboxylate functionalized
School of Chemistry
University of Leeds
Ru(II)(bpy)3 complex capable of binding to cytochrome (cyt) c
and mediating photoreduction.[35] Subsequently our group and
the Ohkanda group designed high affinity Ru(II)(bpy)3
complexes for binding to cyt c and α-chymotrypsin.[36–42] In our
initial study of five different Ru(II)(bpy)3 complexes, two
carboxylic acid functionalized complexes (Figure 1A, complex 1
and 2) were shown to recognize cyt c with nanomolar affinity
and do so selectively over acetylated cyt c and four other
proteins.[36] Complex 2 was also shown to destabilize cyt c.[39]
Analysis of Ru(II)(bpy)3 complexes with 5’-monosubstituted
Woodhouse Lane, Leeds LS2 9JT, UK
E-mail: A.J.Wilson@leeds.ac.uk,
Miss S. H. Hewitt , Dr M. H. Filby, Dr E. Hayes, Dr L. T. Kuhn, Dr A.
P. Kalverda Dr M. E. Webb Prof A. J. Wilson
Astbury Centre For Structural Molecular Biology,
University of Leeds
[b]
Woodhouse Lane, Leeds LS2 9JT, UK
Supporting information for this article is given via a link at the end of the
document.
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Scheme 1. Synthesis of the Ru(II)(bpy)3 complex
bipyridine ligands showed
a
difference in binding affinity
water sensitive acid chloride, with subsequent complexation
using Wilkinson’s reagent.[48] The protected complex formed can
be purified via conventional silica flash column chromatography.
Subsequent deprotection with TFA or LiOH affords complexes 1
and 2 respectively. Deprotection of the larger complex 2 requires
mild conditions and careful reaction monitoring due to the lability
of the anilide bond under both basic and acidic conditions.
between fac and mer isomers (172 nM versus 25 nM for Δ-
isomers respectively), but little difference between Δ and Λ
isomers (25 nM versus 29 nM for mer isomers respectively),
establishing that geometrical shape affects binding.[37] The
Ohkanda group used heteroleptic complexes to propose that
four of the six arms of Ru(II)(bpy)3 complexes bearing bpy
groups with two substituents interact with cyt c.[41] Further
studies have shown that these complexes are able to enter cells,
with little cytotoxicity.[38,41]
Complex 2 inhibits the cyt c/ CCP PPI
These prior strategies employed a rudimentary design that
exploits charge complementarity with the cyt c surface;[36,37,43]
multiple carboxylic acids are present in order to complement
surface exposed basic residues on cyt c. However evidence of
PPI inhibition,[26,44] detailed information on the nature of binding
and any structural information are lacking, which is characteristic
of all but a few studies on protein surface recognition using
classic supramolecular scaffolds.[18,45,46] Inhibited ascorbate
reduction of cyt c,[36,37] is consistent with binding to the CCP
binding site i.e. the haem-exposed edge of cyt c, where there is
a hydrophobic patch surrounded by a ring of basic amino acid
residues.[47] Herein, we show that highly functionalized
Ru(II)(bpy)3 complexes inhibit the cyt c/ CCP interaction and do
so through electrostatically and entropically driven binding of cyt
c in a manner that replicates the binding of cyt c by CCP. Higher
affinity Ru(II)(bpy)3 complexes achieve additional potency
through enthalpic effects. Finally, using high field NMR we
demonstrate that recognition occurs at the haem exposed edge
and hence PPI inhibition is orthosteric. Collectively, this provides
a more rational framework for the design of supramolecular
receptors for cyt c and protein-surfaces more widely.
Figure 2. Complex 2 inhibits the cyt c/CCP PPI (A) luminescence data (λex
=
430 nm, 2 µM ZnCCP (orange), + 2 µM cyt c (pink) shows loss of λmax at 595
nm, + 2 µM cyt c and 4 µM 2 (green) shows recovery of λmax at 595 nM and
reduced λmax at 625 nM relative to 4 µM 2 alone (blue)
Given that the affinity of complex 2 for cyt c which we previously
reported,[36] is greater than that of CCP for cyt c[49] we
anticipated 2 would be a potent inhibitor of the cyt c/ CCP
interaction. A luminescence quenching assay was implemented
(Fig. 2) wherein the luminescence emission from Zn-
protoporphyrin substituted CCP[50] is first quenched upon
interaction with cyt c and then recovered upon displacement with
the ruthenium complex. Signal overlap with the Ru(II)(bpy)3
luminescence (λmax~625 nm) complicates interpretation,
however simultaneous loss of MLCT luminescence is observed
Results and Discussion
Synthesis
Ru(II)(bpy)3 synthesis proceeds via the route shown in Scheme
1, using a tert-butyl ester or methyl ester protecting group
strategy for complex 1 or 2 respectively. In this generic route,
the ligand is first assembled by amide bond formation, via a
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relative to the complex in the absence of cyt c. A native agarose
making the assumption that ΔH and ΔS are temperature-
gel supports successful PPI inhibition (see ESI, Fig. S1).
independent
Eq. 1
Binding is Entropically favourable and electrostatic in
nature.
These data show that for complex 1 binding to cyt c is
primarily driven by entropic contributions with a small favorable
enthalpic contribution, whereas for complex
2 it is both
entropically and enthalpically driven. In comparison the cyt c/
CCP interaction is entropically driven, and enthalpically is mildly
unfavourable.[49] Thus, complex 1, with fewer carboxylates, more
closely matches the thermodynamic profile of CCP in binding to
cyt c. A plausible hypothesis for enhanced binding of complex 2
to cyt c, is that the additional carboxylic acids form increased
numbers of salt bridges with the basic amino acids on the cyt c
surface.
Table 1. Thermodynamic parameters derived from the van’t Hoff analysis for
the binding of complexes 1 and 2 to cyt c (errors derived from triplicate
experiments), and literature values for the cyt c/CCP interaction under similar
conditions[49]
Complex 1
-6.6 ± 0.4
24.5 ± 0.4
-31.0 ± 0.4
Complex 2
-26.3 ± 3.0
16.0 ± 3.0
-42.3 ± 0.0
CCP[49]
ΔH / kJ mol-1
TΔS (25 °C) / kJ mol-1
ΔG (25 °C) / kJ mol-1
9.4 ± 0.8
38.4 ± 0.9
-27.9 ± 1.0
Table 2. Binding in variable ionic strengths, 5 mM sodium phosphate, 0.2 mg
mL-1 BSA, pH 7.5, variable concentration NaCl, nd=not determined
Ionic strength/ mM
Complex 1 Kd / μM
2.88 ± 0.46
4.25 ± 0.47
10.30 ± 1.61
20.23 ± 0.16
nd
Complex 2 Kd/ nM
25.3 ± 2.4
8.39
13.39
18.39
28.39
48.39
64.8 ± 13.7
196.5 ± 59.2
426.5 ± 59.8
2040.9 ± 152.6
To further understand the electrostatic contribution to
binding, affinities were determined at different ionic strength (I).
Cyt c binding by both complex 1 and 2 is highly dependent upon
ionic strength (Table 2), with binding affinity decreasing on
increasing ionic strength, suggesting electrostatics dominate
binding. The Kd values could be fit to the Debye-Hückel
relationship (Eq. 2) (Figure 3(B)), in this case using a Güntelberg
approximation (Eq. 3), which is valid up to I = 100 mM.
Figure 3. Van’t Hoff and Debye-Hückel analysis on the binding interactions
between cyt c and complexes 1 and 2, (a) Representative van’t Hoff analysis,
binding in 5 mM sodium phosphate, 0.2 mg mL-1 BSA, pH 7.5, temperature
range 25 to 45 °C, (errors in curve fitting for a single replicate are shown) (b)
Debye-Hückel analysis, using the Güntelberg approximation, binding in 5 mM
sodium phosphate, 0.2 mg mL-1 BSA, pH 7.5, and variable concentrations
NaCl, (variation in Kd from two replicates is shown)
Eq. 2
Eq. 3
The binding affinities of complexes 1 and 2 towards cyt c were
measured using a luminescence quenching assay,[36] where the
luminescence of the ruthenium complexes is quenched on
binding to cyt c through photoinduced electron transfer to its
haem group. Previously, cuvette-based fluorescence was used
for binding studies,[36,37] however optimization of the assay on a
384 well plate was required for higher-throughput screening of
the binding under different conditions. Addition of a blocking
agent, bovine serum albumin (BSA), was found to be required to
allow for agreement between the two methods. The addition of
BSA accompanied a concurrent decrease in binding affinity
(from Kd 10.5 ± 0.4 nM to 42.9 ± 3.1 nM for complex 2) (See ESI
Fig. S1). Determination of Kd at different temperatures and
subsequent van’t Hoff analyses (Figure 3A) provided
thermodynamic parameters (Table 1) for binding (Equation 1),
0
From this relationship the parameters Kd and Z1Z2 can be
established (Table 3), providing an estimate of the affinity at I =
0 and the product of the interacting positive and negative
charges. The data were consistent with the Güntelberg
approximation for both complexes (Figure 3B), and the
calculated values of Kd0 show high affinity binding for complex 2
and weaker binding for complex 1 at zero ionic strength. The
product, Z1Z2, provides an indication of the charges involved in
the interaction, with complex 2 having a larger value than
complex 1 and CCP. Using these data, the charge on the
complex interacting with cyt c can be estimated. A rudimentary
interpretation of this date is made possible by assuming that cyt
c has the same charge in all cases (calculated to be ~6 at pH
7.5);[51] the charge on complex 1, 2 and CCP can thus be
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calculated as 4.3, 5.9 and 4.8, respectively. Complex 1 and CCP
have relatively similar charges, suggesting they make similar
electrostatic interactions with cyt c. Complex 2 has a larger
charge indicating increased electrostatic interactions with cyt c.
This is consistent with the van’t Hoff analyses. Accounting for
the crudeness of the Debye-Hückel approximation where small
(~3 Å), evenly dispersed charges are assumed (even when
using the Güntelberg extension), the data indicate that perhaps
not all carboxylates are deprotonated under the assay conditions
(i.e. pH 7.4) and/or that a limited number of carboxylates are
needed for productive protein surface recognition (even fewer
than the number identified in the “deletion” study by the
Ohkanda group using heteroleptic complexes),[41]
hence the pH of the solution was expected to affect recognition
of cyt c by Ru(II)(bpy)3 complexes. To investigate the binding
affinity between complex 2 and cyt c over a broad pH regime,
btp was used as it allows for a pH range of 6.5–9.5. The affinity
follows an inverted bell shaped profile (Figure 4A), which maps
reasonably well onto the ionization state of cyt c (Figure 4A
inset).[53] The affinity between pH 7.0–8.5 is relatively constant
with decreased binding observed at pH 6.5 and pH 9.0.
Residues that become protonated/deprotonated in this pH
regime are His-33 and Lys-79 respectively.[53] Lys-79 (green) is
at the haem exposed edge (Figure 4B) where binding of
complex 2 is thought to occur, whereas His-33 (pink) is on the
distal face of cyt c. A number of reasons for a decrease in
binding affinity at this pH are possible: (a) complex 2 binds to a
different or multiple sites on cyt c, (b) binding of complex 2
causes subtle conformational changes that transmit to the distal
face of cyt c affecting the pKa of His-33, (c) protonation of His-33
causes subtle conformational changes that affect binding
interactions on the haem exposed edge, or (d) the protonation
state of complex 2 is changed at pH 6.5. More careful analysis
of the pH-Kd/ionization state profiles reveals a discrepancy. The
ionization state of cyt c drops at pH 8.0 rather than pH 8.5 where
the binding diminishes, suggesting that binding of complex 2
might mask Lys-79 and increase its pKa. In contrast there is no
difference in the profiles for Kd and ionization state of cyt c in the
lower pH range suggesting that the pKa of His-33 is not affected
by binding and that loss of affinity more likely derives from a
change in ionization state on complex 2.
Table 3. Parameters derived from the Güntelberg approximation of Debye-
Hückel analysis for the binding of complexes 1 and 2 to cyt c and literature
values for CCP in similar conditions[52]
Complex 1
253 ± 5
Complex 2
1.11 ± 0.21
35.6 ± 1.3
CCP[52]
Kd0 / nM
Z1Z2
40.7 ± 23.0
28.8 ± 4.8
25.9 ± 1.9
Table 4. Binding affinities for complex 2 to cyt c in different buffers. All
buffers were at 5 mM concentration, pH 7.5
Buffer
Sodium phosphate
Potassium phosphate
MOPS
Kd / nM
42.9 ± 3.1
26.2 ± 3.1
35.2 ± 3.1
31.2 ± 3.1
106.3 ± 32.6
133.5 ± 37.4
HEPES
Tris
btp
Differences in affinity between cyt c and complex 2 were
also studied in different buffers (Table 4). Variation in affinity
might discriminate different contributions to binding as negatively
charged anions must be displaced from cyt c and positively
charged cations from complex 2. In potassium and sodium
phosphate no difference in affinity between complex 2 and cyt c
is observed, indicating interaction of the cationic buffer
components with complex 2 are not significant. For binding of
cyt c to complex 2 in phosphate or sulfonic acid buffers (MOPS
and HEPES), similar affinities are also observed. This suggests
the nature of the anion and, more importantly, hydrophobicity of
the buffer are not significant in mediating molecular recognition,
and, reinforce the conclusions gleaned from Debye-Hückel
analysis that the interaction is dominated by electrostatic
contributions. For the tris buffers (btp and tris) a small decrease
in binding affinity is observed. Although a difference in behavior
due to the counter chloride anion cannot be excluded, this may
be due to the ability of btp and tris to make different interactions
with both cyt c and complex 2; in addition to the ammonium
function, the hydroxyls on the buffer may make chelating
hydrogen-bonds with charged residues on either.
Figure 4. Effect of pH on the binding of complex 2 to cyt c. (a) Binding affinity
over the range pH 6.5 – 9.0, inset the electrostatic interaction factor (ω) of cyt
c over a range of pHs (base limb of titration curve)[53], (b) Cyt c structure (PDB
ID 1U75)[54] with residues that become protonated at pH 6.5 and 9.0, His-33
(pink) and Lys-79 (green) respectively
Cyt c is a stable protein that does not unfold over a wide
range of pHs, however its ionization state is affected by pH,[53]
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Figure 5. 1H-15N HSQC NMR data of complex 1 binding to cyt c. (A) Region of the overlaid HSQC spectrum of cyt c (red) and cyt c with 0.5 eq complex 1 (blue),
Inset shows zoom in of part of the spectrum, showing some peaks staying the same, some having shifted and one disappearing. (B) 1H-15N chemical shift
differences () for the different amino acid residues with and without complex 1. Gaps are for prolines and unassigned amino acids, red bars show amino acids
for which the signal disappears due to significant line-broadening of NH cross-peaks on addition of complex 1. (C) Chemical shift perturbation map of cyt c,
molecular surface of cyt c generated from PyMol (PDB ID 1U75),[54] with coloring corresponding to the extent of chemical shift changes () on addition of the
complex. Amino acids with 15N-1H resonances that disappear in dark red, exhibit large chemical shift changes (Δδ>0.03) in red, moderate changes (Δδ>0.02) in
orange, small changes (Δδ>0.015) in yellow-orange and very small chemical shift changes (Δδ>0.01) in yellow. (D) perturbation map of cyt c (as(C)) in complex
with CCP (purple), this view corresponds to that of the central top image in Figure 5C, (PDB ID 1U75)[34]
complex 1, the NMR data show several cross-peaks having
disappeared, while others display chemical shift changes
High-Field NMR reveals complexes 1 and 2 bind to the CCP
binding site on cyt c
ranging from 0.015-0.05 ppm indicating the presence of protein-
ligand interactions (Figure 5A and 5B). When these chemical
shift changes are mapped onto the structure of cyt c from the cyt
c/ CCP crystal structure,[54] the data indicate binding occurs
predominantly to one side of the haem group, with the opposite
face having very few amino acids with sizeable shifts in their
HSQC peaks (Figure 5C). The binding site is in a similar location
to that of carboxylic acid functionalized porphyrins.[18] In
comparison to the cyt c/ CCP interaction (Figure 5D), it can be
seen that the amino acids whose cross-peaks have shifted are
in and around the PPI interface, indicating complex 1 is an
effective mimic of CCP, binding at the same face and capable of
acting as an orthosteric inhibitor of the interaction.
While the pH data provide some crude structural information on
the cyt c binding site of Ru(II)(bpy)3 complexes, more detailed
residue-specific, atomic-level data were sought. To identify the
binding site of complex 1 and 2 on cyt c, a sensitivity-enhanced
natural abundance 1H-15N HSQC spectrum of cyt c in the
presence and absence of complex 1 was recorded, using a 950
MHz NMR spectrometer. 2 mM sodium ascorbate was added to
the buffer, to reduce the iron in cyt c from paramagnetic Fe(III) to
diamagnetic Fe(II), thus minimizing its influence on the
spectrum, i.e. paramagentic line broadening. The binding of the
complexes to cyt c for reduced versus oxidized cyt c is similar
(for complex 2, Kd = 92.4 ± 5.5 and 49.6 ± 13.3 nM respectively,
in 5 mM phosphate, 2 mM sodium ascorbate, 0.2 mg mL-1 BSA).
The assignment of the 1H-15N HSQC spectrum of horse
heart cyt c has previously been obtained.[55] Upon addition of
Attempts to acquire data in the presence of complex 2
were difficult due to the high affinity binding and the relatively
high concentrations required for natural abundance NMR. At 1:1
ratios of cyt c and complex 2, data could not be obtained due to
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solid. The dry acid chloride was then redissolved in dry chloroform (20
mL) and added dropwise to a stirred solution of di-tert butyl L-aspartic
acid.HCl (253 mg, 0.901 mmol) and triethylamine (0.25 mL, 1.80 mmol)
in dry chloroform at 0°C. The reaction mixture was warmed to room
temperature and refluxed for 48 hours. The mixture was cooled to room
temperature and the solvent removed to yield the crude product as a
brown oil. This was purified by flash column chromatography (3 % - 6 %
MeOH in CHCl3) to yield the product as a yellow solid (262 mg, 0.375
mmol, 91 %); 1H NMR (300 MHz, CDCl3) δ ppm 1.49 (s, 18 H, H1/H2),
1.52 (s, 18 H), 2.91 (dd, J=17.2, 4.3 Hz, 2 H), 3.04 (m, J=17.2, 4.3 Hz,
2H), 4.92 (dt, J=7.5, 4.3 Hz, 2 H), 7.46 (d, J=7.5 Hz, 2 H), 7.77 (dd,
J=5.0, 1.65 Hz, 2 H), 8.78 (app. s, 2 H), 8.83 (d, J=5.0 Hz, 2 H); 13C NMR
(126 MHz, CDCl3) δ ppm 28.0, 28.1, 37.5, 49.7, 81.9, 82.8, 118.0, 121.8,
142.3, 150.1, 156.3, 165.1, 169.5, 170.2 ; IR (solid state) 3346, 2975,
2928, 1723, 1650; ESI-HRMS: found m/z 699.3615, [C36H51N4O10]+
requires 699.3599
the formation of oligomers and a concomitant loss of NMR signal
intensity, caused by significant line-broadening.This unsurprising
to us as given the potential for aggregation at higher
concentrations and the observation of additional binding modes
in NMR studies using porphyrins.[18] Further evidence of an
additional/alternative binding mode for the larger complex 2, is
the visualization of a second binding event for complex 2 with
yeast cyt c, but a single binding event for complex 1 (See ESI,
Fig. S2). Even at 1:2 equivalents complex 2:cyt c multiple
signals disappeared, so detailed information as to the binding
site could not be gleaned, however of the signals present,
chemical shift changes were detected for regions of the protein
backbone located on the same binding face as for complex 1,
and on the haem exposed edge.
Tris
((2S,2’R)-tetra-tert-butyl-2,2’-(([2,2’-bipyridine]-4,4’-
dicarbonyl)bis (azanediyl))disuccinate) ruthenium(II) dinitrate
Conclusions
(2S,2'R)-tetra-tert-butyl-2,2'-(([2,2'-bipyridine]-4,4'-dicarbonyl)bis
We have performed a detailed study on the cyt c binding
properties of two synthetic Ru(II)(bpy)3 complexes 1 and 2. The
ruthenium complexes are potent ligands for selective protein
surface recognition of cyt c and capable of inhibiting the cyt c/
CCP PPI. Binding is shown to be entropically favorable and
driven by complementary electrostatic interactions between the
basic protein and acidic Ru(II)(bpy)3 complexes. This profile is
consistent with accurate mimicry of the cyt c binding properties
of CCP. Higher-affinity recognition of the protein target can be
achieved through the addition of further acidic motifs on the
Ru(II)(bpy)3 complexes allowing additional enthalpically
favorable electrostatic interactions to occur. Finally, NMR
experiments establish that the Ru(II)(bpy)3 complexes 1 and 2
bind to the solvent exposed cyt c surface further underscoring
the ability of the complexes to act as mimics of CCP and
confirming an orthosteric mode of PPI inhibition. These studies
highlight the value of detailed analyses of protein-surface
recognition by supramolecular hosts in terms of rationalizing
structure function relationships, and informing subsequent
designs. Moreover the conclusions of this study point to a future
need for syntheses/ assembly of asymmetrically functionalized
Ru(II)(bpy)3 complexes to maximize productive protein-ligand
contacts and selectivity of protein surface recognition. This and
the application of our approach to therapeutically attractive
protein targets will form the basis of future studies by our group.
(azanediyl))
disuccinate
(300
mg,
0.429
mmol),
(dimethylsulfoxide)dichlororuthenium (II) (65 mg, 0.134 mmol), silver
nitrate (46 mg, 0.268 mmol) and ethanol (20 mL) were heated under
reflux for 7 days. After which time the reaction mixture was filtered hot
and concentrated. The red solid was then loaded onto an SP Sephadex
column and eluted with 1:1 acetone: 0.1 M NaCl solution and all the red
fractions collected and concentrated. The combined red fractions were
redissolved in acetone and filtered to remove sodium chloride, and this
was repeated until no more white salt was visible in the concentrated
sample. The complex was then purified by flash chromatography (1 – 3
% MeOH in CHCl3) and the red fractions collected. These were
concentrated, redissolved in CHCl3 and extracted with water to yield the
product as a red solid (77 mg, 0.034 mmol, 25 %); 1H NMR (300 MHz,
Acetone) δ ppm 2.10 (app s, 108 H), 2.81 - 3.07 (m, 24 H), 4.79 - 5.07
(m, 24 H), 7.89 (dd, J = 15.8, 6.6 Hz, 12 H), 8.37 (dd, J = 15.8, 8.5 Hz, 12
H), 8.82 - 8.98 (m, 12 H); 13C NMR (126 MHz, CDCl3) δ ppm 27.9, 28.1,
29.6, 37.1, 50.9, 81.4, 82.4, 123.5, 143.5, 157.2, 157.3, 162.2, 169.2,
169.4, 170.0; ESI-HRMS: found m/z 1098.4854, [C108H150N12O30Ru]2+
requires 1098.4822; λmax (MeOH): 306 nM (ε/ dm3 mol-1 cm-1 240 723
981)
Tris
(2S,2'R)-2,2'-(([2,2'-bipyridine]-4,4'-dicarbonyl)bis
(azanediyl))disuccinicacid) ruthenium(II) ditrifluoroacetate, complex
1
Tris ((2S,2'R)-tetra-tert-butyl -2,2'-(([2,2'-bipyridine] -4,4'-dicarbonyl)
bis(azanediyl)) disuccinate) ruthenium(II) dinitrate(68 mg, ), TFA (4.5 mL)
and water (0.5 mL) were stirred for 3 days. The reaction mixture was
then concentrated in vacuo to yield the product as a red-black solid (57
1
mg, 0.0294 mmol, 98 %); H NMR (500 MHz, D2O) δ ppm 8.97 (s, 1 H),
Experimental Section
7.90 (s, 1 H), 7.70 (s, 1 H), 4.61 (s, 1 H), 2.78 (s, 1 H), 2.67 (s, 1 H’), ; IR
(solid state) 3182, 3050, 1648; ESI-HRMS: found m/z 762.1081,
[C60H54N12O30Ru]2+ requires 762.1056;
Synthesis
Synthesis was adapted from literature.[36] A representative synthesis of
complex 1 is shown below. The synthesis of complex 2 is described in
the supplementary information.
Protein Expression and Purification
Cytochrome c peroxidase (CCP) was overexpressed in Escherichia coli
(E. coli) using the plasmid pT7CCP) in which expression was placed
under the control of T7 RNA polymerase. The enzyme was isolated from
E. coli BL21 (DE3) as an apo-enzyme which was purified according to
the literature.[1]. A 2 L culture of the expression strain supplemented with
ampicillin was grown at 37 °C for 36 h in a medium containing (per litre)
10 g of bactotryptone, 8 g of yeast extract, 5 g of NaCl, 1 mL of glycerol,
and 100 mg of ampicillin. Subsequent steps were performed at 4 °C. The
cells were harvested by centrifugation at 6000g for 10 min, resuspended
(2S,2'R)-tetra-tert-butyl-2,2'-(([2,2'-bipyridine]-4,4'-dicarbonyl)bis
(azanediyl)) disuccinate
2,2’-bipyridine-4,4’-dicarboxylic acid (100 mg, 0.400 mmol), triethylamine
(1 drop) and thionyl chloride (4 mL) were heated under reflux for 16
hours. The mixture was cooled to room temperature and the thionyl
chloride removed in vacuo to yield the acid chloride as an orange-red
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in 40 mL of buffer containing 200 mM potassium phosphate pH 7.5, 2
tablets of Roche protease inhibitor tablets mini, and 1 mM EDTA, and
lysed by passing through a cell disrupter. The lysate was diluted with 100
mL of cold H2O. Enough ascorbic acid was added to bring the buffer to
5mM. To improve the ratio of the soret band to the band at 280 nm of
FeCCP, an excess of haem was added, 80 mg of haemin/ 12 L culture
was dissolved in a minimal amount of 100 mM KOH in the dark, and
diluted 10 times with 100 mM potassium phosphate, pH 6. The haem
solution was gradually added to clarified lysate on ice over 30 minutes
with gentle stirring, then stirred on ice for 1 hour in the dark. The excess
haem was then precipitated by first acidifying the solution with 100 mM
acetic acid to pH 5.0, and freezing the solution in dry ice until just frozen.
The solution was then allowed to just thaw with gentle shaking at 37 °C
The solution was centrifuged at 10 000 – 12 000 rpm for 20 minutes and
the supernatant decanted. The clear supernatant was loaded onto a
DEAE-Sepharose CL-6B (3 x 5 cm) column equilibrated with 50 mM
potassium phosphate, pH 6, and washed with the same buffer. After
elution with 500 mM potassium phosphate, pH 6, the enzyme-containing
fractions were diluted with an equal volume of cold H2O and
concentrated to approximately 1 mL by ultrafiltration (Amicon YM-10
membrane). The sample was centrifuged at 12000g for 2 min to remove
insoluble material, loaded onto a Sephadex G-75 superfine column (3 x
60 cm) and eluted with 100 mM potassium phosphate, pH 6, and 1 mM
EDTA. The fractions containing A408/A280 >1.1 were pooled (protein
concentration was determined from the molar absorptivity; ε = 55 mM-1
cm-1, at 282 nm).
Luminescence quenching assays
All stocks for luminescence intensity assays were made up in 5 mM
phosphate buffer (pH 7.5). Ruthenium complex stocks were made up to
2 mM. Horse heart and yeast cyt c was obtained from Sigma Aldrich, and
used without further purification. A cytochrome c stock was made up to
~1 mM, and the concentration accurately determined using the molar
extinction coefficient at 550 nm of 2.95 × 104 mol-1 dm3 cm-1 for horse
[56]
heart cyt c
and 2.11 × 104 mol-1 dm3 cm-1 for yeast cyt c[56] after
reduction by addition of one microspatula of sodium dithionite. Assays
with oxidized cyt c in ascorbate containing buffer used cyt c oxidized with
K3Fe(CN)6 followed by dialysis into 5 mM sodium phosphate, 2 mM
sodium ascorbate, pH 7.5 buffer, to remove the excess K3(CN)6. The
concentration of oxidized cyt c was determined by using the molar
extinction coefficient at 410 nm of 1.061 × 105 mol-1 dm3 cm-1[57].All
buffers used were at 5 mM concentration, 0.2 mg mL-1 BSA, pH 7.5,
unless otherwise stated.
Fluorometer luminescence quenching assays were measured on a Jobin-
Yvon Spex Fluorolog-3 fluorometer. Measurements were taken in a 4 mL
quartz cuvette with excitation at 467 nm and emission measured over the
range 575 – 675 nm, with 10 nm slit widths on both excitation and
emission. Peak maxima were recorded over the entire cyt
concentration gradient.
c
Plate reader luminescence quenching assays, were performed using a
Perkin Elmer EnVisionTM 2103 MultiLabel plate reader, with excitation at
467 nm, and emission at 630 nm fixed wavelength. A 2/3 dilution regime
in a 384 well plate (Optiplate) was used (total well volume 50 µL), with
each result measured in triplicate. The Kd ranges possible for this assay
is ~5 nM – ~100 μM
In order to exchange the haem for Zn porphyrin, the haem was removed
using the acid butanone method[2] with minor modifications. A ~1 mM
solution of haem CCP in 100 mM potassium phosphate buffer was
diluted with 4 volumes of ice-cold water. The CCP solution was brought
to 100 mM fluoride by addition of 1 M KF solution, breaking the haem-
protein linkage and turning the solution green. The haem was removed
by lowering the pH of the solution to pH 3.2-3.3 by adding ice cold 0.1 M
HCl dropwise, with gentle stirring. The haem was then extracted by
adding an equal volume of ice cold 2-butanone, shaking for 30 seconds
and centrifuged for 1 minute at 1000g. The brown layer was siphoned
away and the extraction repeated until the aqueous layer became
colourless. The resulting apoCCP solution was diluted with a half volume
of cold water and dialysed against 2-3 changes of 10 mM NaHCO3
solution. It was then dialysed against water, changing the outer solution
every 2 hours until the bag no longer smelt of butanone (~24 hours),
followed by dialysis into 10 mM Tris-HCl, pH 7. A 4:1 excess of porphyrin
was dissolved in 200-500 μL of 100 mM KOH and diluted 5-10 times with
water. The porphyrin solution was added to the protein solution, and the
protein solution titrated to pH 7.8 with 100 mM KOH. In the dark, the
alkaline porphyrin solution was added dropwise with gentle stirring to
apoCCP until ~2 fold excess of porphyrin was present. The solution was
left to stand at near pH 8 for 20-30 minutes than brought to pH 6.5-7.0 by
addition of 1 M monobasic potassium phosphate. The protein was
exchange into 25 mM potassium phosphate, pH 6.5 and concentrated by
ultracentrifugation to 0.5-1.0 mM CPP. The protein was loaded onto a
small column of DEAE Sepharose CL-6B, pre-equilibrated with 25 mM
potassium phosphate, pH 6.5. The column was rinsed with around half a
volume of loading buffer, and the metalloporphyrin CCP eluted with 0.6 M
potassium phosphate, pH 6.5.
In all assays the ruthenium complex concentration was kept constant,
with the concentration of cyt c being varied through the assay, as
described below. Results obtained were fitted, using Origin9, to a 1:1
binding isotherm:
Where I = change in relative luminescence intensity (I/I0), m = maximum
value of I, a = concentration of complex, K = dissociation constant, b =
concentration of protein added
Protein NMR
1
Sensitivity enhanced H-15N HSQC NMR correlation spectra of ligand-
bound and unbound forms of cyt c, purchased from Sigma Aldrich, were
carried out at natural abundance using a 950 MHz Bruker AscendTM
Aeon spectrometer operating at a proton (1H) resonance frequency of
950.13 MHz equipped with a Bruker TCI triple-resonance cryo-probe.
NMR acquisitions were carried out in 5 mM sodium phosphate, 2 mM
sodium ascorbate, pH 7.25 buffer. For cyt c alone, spectra were taken at
2 mM protein concentration. With complex 1, 1 mM cyt c and 0.5 mM
complex 1 were used, to a total volume of 600 μL. Spectra were
analysed using the CcpNmr Analysis software package and the chemical
shift perturbations were calculated as the square root of the sum of the
isotope weighted shift differences squared (Eq. 4),
Inhibition of Cyt c/CCP by fluorescence recovery
To a 500 µL micro fluorescence cell (Hellma Analytics) containing 500 µL
of 2 µM ZnCCP (280 = 55 mM-1cm-1) was added 1 eq of cytochrome c, in
a solution containing 2 μM ZnCCP. 2 equivalents of complex 2 were then
added. Fluorescence spectra where taken at each point (ex. 430 nm). A
separate comparative spectra for complex 2 was taken using identical
instrument settings
where Δδ is the overall change in chemical shift, ΔδN is the change in the
nitrogen dimension and ΔδH is the change in the proton dimension,
respectively. The change in the proton dimension is scaled by the ratio of
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the gyromagnetic ratio of 15N (γN) and 1H (γH) to account for the larger
Chem. Soc. 2014, 136, 2008–2016.
chemical shift range of nitrogen.
[25] D. Grünstein, M. Maglinao, R. Kikkeri, M. Collot, K. Barylyuk, B. Lepenies,
F. Kamena, R. Zenobi, P. H. Seeberger, J. Am. Chem. Soc. 2011, 133,
13957–13966.
Keywords: Protein-protein interactions • receptors •
supramolecular chemistry • molecular recognition • protein
surface recognition
[26] H. Bayraktar, P. S. Ghosh, V. M. Rotello, M. J. Knapp, Chem. Commun.
(Camb). 2006, 1390–2.
[27] K. Saha, S. S. Agasti, C. Kim, X. Li, V. M. Rotello, Chem. Rev. 2012, 112,
2739–79.
[28] †,§ Nicholas O. Fischer, § Ayush Verma, § Catherine M. Goodman, § and
Joseph M. Simard, †,§ Vincent M. Rotello*, 2003, DOI
10.1021/JA0352505.
Acknowledgments
[29] T. Schrader, S. Koch, Mol. Biosyst. 2007, 3, 241–8.
[30] C. Renner, J. Piehler, T. Schrader, J. Am. Chem. Soc. 2006, 128, 620–
628.
The authors wish to Prof Mike Knapp (UMass, Amherst) for the
gift of the CCP plasmid and Taylor Page (Prof. Brian Hoffman’s
group of Northwestern University) for sharing the modified
purification procedure. This work was supported by the
Engineering and Physical Sciences Research Council
[31] Reza Zadmard, T. Schrader*, J. Am. Chem. Soc. 2004, 127, 904–915.
[32] A. J. Wilson, J. Hong, S. Fletcher, A. D. Hamilton, Org. Biomol. Chem.
2007, 5, 276–85.
[33] M. Jewginski, L. Fischer, C. Colombo, I. Huc, C. D. Mackereth,
ChemBioChem 2016, 17, 727–736.
[EP/L504993/1,
EP/KO39292/1] and The Wellcome Trust [108466/Z/15/Z].
EP/F039069/1,
EP/F038712/1
and
[34] S. A. Kang, P. J. Marjavaara, B. R. Crane, J. Am. Chem. Soc. 2004, 126,
10836–10837.
[1] M. R. Arkin, J. A. Wells, Nat. Rev. Drug Discov. 2004, 3, 301–17.
[2] A. J. Wilson, Chem. Soc. Rev. 2009, 38, 3289–300.
[35] H. Takashima, S. Shinkai, I. Hamachi, Chem. Commun. 1999, 2345–
2346.
[3] F. N. B. Edfeldt, R. H. A. Folmer, A. L. Breeze, Drug Discov. Today 2011,
16, 284–287.
[36] J. Muldoon, A. E. Ashcroft, A. J. Wilson, Chem. A Eur. J. 2010, 16, 100–
3.
[4] M. J. Waring, J. Arrowsmith, A. R. Leach, P. D. Leeson, S. Mandrell, R.
M. Owen, G. Pairaudeau, W. D. Pennie, S. D. Pickett, J. Wang, et al.,
Nat. Rev. Drug Discov. 2015, 14, 475–486.
[37] M. H. Filby, J. Muldoon, S. Dabb, N. C. Fletcher, A. E. Ashcroft, A. J.
Wilson, Chem. Commun. 2011, 47, 559–61.
[38] S. J. Turrell, M. H. Filby, A. Whitehouse, A. J. Wilson, Bioorg. Med. Chem.
Lett. 2012, 22, 985–8.
[5] V. Azzarito, K. Long, N. S. Murphy, A. J. Wilson, Nat. Chem. 2013, 5,
161–173.
[39] A. J. Wilson, J. R. Ault, M. H. Filby, H. I. A. Philips, A. E. Ashcroft, N. C.
Fletcher, Org. Biomol. Chem. 2013, 11, 2206–12.
[40] J. Ohkanda, R. Satoh, N. Kato, Chem. Commun. 2009, 6949–51.
[41] Y. Yamaguchi, N. Kato, H. Azuma, T. Nagasaki, J. Ohkanda, Bioorg.
Med. Chem. Lett. 2012, 22, 2354–8.
[6] M. R. Arkin, A. Whitty, Curr. Opin. Chem. Biol. 2009, 13, 284–290.
[7] D. a Uhlenheuer, K. Petkau, L. Brunsveld, Chem. Soc. Rev. 2010, 39,
2817–2826.
[8] S. Fletcher, A. D. Hamilton, Curr. Opin. Chem. Biol. 2005, 9, 632–8.
[9] S. H. Hewitt, A. J. Wilson, Chem. Commun. 2016, 52, 9745–9756.
[10] L. Milroy, T. N. Grossmann, S. Hennig, L. Brunsveld, C. Ottmann, Chem.
Rev. 2014, 114, 4695–4748.
[42] J. Ohkanda, Chem. Rec. 2013, 13, 561–75.
[43] S. Shinoda, H. Tsukube, Chem. Sci. 2011, 2, 2301.
[44] Y. Wei, G. L. McLendon, M. A. Case, C. B. Purring, T. Yu, A. D. Hamilton,
Q. Lin, H. S. Park, C.-S. Lee, Chem. Commun. 2001, 1580–1581.
[45] M. Mallon, S. Dutt, T. Schrader, P. B. Crowley, ChemBioChem 2016, 17,
774–783.
[11] R. Zadmard, N. S. Alavijeh, RSC Adv. 2014, 4, 41529–41542.
[12] M. A. Blaskovich, Q. Lin, F. L. Delarue, J. Sun, H. S. Park, D. Coppola, A.
D. Hamilton, S. M. Sebti, Nat. Biotechnol. 2000, 18, 1065–70.
[13] H. S. Park, Q. Lin, A. D. Hamilton, J. Am. Chem. Soc. 1999, 121, 8–13.
[14] Y. Hamuro, M. C. Calama, H. S. Park, A. D. Hamilton, Angew. Chemie
Int. Ed. English 1997, 36, 2680–2683.
[46] Ayush Verma, and Joseph M. Simard, V. M. Rotello*, Langmuir 2004, 20,
4178–4181.
[47] H. Pelletier, J. Kraut, Science (80-. ). 1992, 258, 1748–1755.
[48] A. Spencer, I. Evans, A. Spencer, G. Wilkinson, J.C.S. Dalt. 1973, 204–
209.
[15] R. E. McGovern, H. Fernandes, A. R. Khan, N. P. Power, P. B. Crowley,
Nat. Chem. 2012, 4, 527–33.
[16] D. Margulies, Y. Opatowsky, S. Fletcher, I. Saraogi, L. K. Tsou, S. Saha,
I. Lax, J. Schlessinger, A. D. Hamilton, Chembiochem 2009, 10, 1955–8.
[17] H. Zhou, L. Baldini, J. Hong, A. J. Wilson, A. D. Hamilton, J. Am. Chem.
Soc. 2006, 128, 2421–5.
[49] J. E. Erman, G. C. Kresheck, L. B. Vitello, M. A. Miller, Biochemistry 1997,
36, 4054–4060.
[50] T. Y. Yonetani, TEE J. Biol. Chem. 1967, 242, 5008–5013.
[51] H. B. Bull, K. Breese, Biochem. Biophys. Res. Commun. 1966, 24, 74–78.
[52] L. B. Vitello, J. E. Erman, Arch. Biochem. Biophys. 1987, 258, 621–629.
[53] J. L. Res, B. B. Acta, B. B. Acta, B. B. Acta, Biochemistry 1976, 15, 1909–
1914.
[18] P. B. Crowley, P. Ganji, H. Ibrahim, ChemBioChem 2008, 9, 1029–33.
[19] S. Mignani, S. El Kazzouli, M. M. Bousmina, J.-P. Majoral, Chem. Rev.
2014, 114, 1327–42.
[20] F. Chiba, T. Hu, L. J. Twyman, M. Wagstaff, Chem. Commun. 2008,
4351–4353, 4351–4353.
[54] G. W. Bushell, G. V Louie, G. D. Brayer, J. Mol. Biol. 1990, 214, 585–595.
[55] W. Liu, J. Rumbley, S. W. Englander, a J. Wand, Protein Sci. 2003, 12,
2104–2108.
[21] H. Azuma, Y. Yoshida, D. Paul, S. Shinoda, H. Tsukube, T. Nagasaki,
Org. Biomol. Chem. 2009, 7, 1700–1704.
[56] B. F. Van Gelder, E. C. Slater, The Extinction Coefficient of Cytochrome
c, Elsevier, 1962.
[22] D. Paul, H. Miyake, S. Shinoda, H. Tsukube, Chem. - A Eur. J. 2006, 12,
1328–38.
[57] E. Margoliash, N. Frohwirt, U. W. Riley, D. P. Phil, Tran8, 409 247a, S.
Blackburn, A. G. Lowther, R. Cecil, A. G. Ogston, et al., Biochem. J. 1959,
71, 570–572.
[23] J. Simpson, Y. Liu, W. A. Goddard, J. Giri, M. S. Diallo, ACS
Nanotechnol. 2011, 5, 3456–3468.
[24] D. Ponader, P. Maffre, J. Aretz, D. Pussak, N. M. Ninnemann, S. Schmidt,
P. H. Seeberger, C. Rademacher, G. U. Nienhaus, L. Hartmann, J. Am.
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Entry for the Table of Contents (Please choose one layout)
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Sarah H. Hewitt, Maria H. Filby, Ed
Hayes, Lars T. Kuhn, Arnout P.
Kalverda, Michael E. Webb and Andrew
J. Wilson*
Page No. – Page No.
Protein surface mimetics;
understanding how ruthenium
tris(bipyridines) interact with proteins
Ruthenium
tris(bipyridines)
binding to cyt c acting as protein surface mimetics, including PPI inhibitions, further
understanding of the binding interaction by looking at binding in many variable
conditions, and NMR to probe where the binding site on cyt c is.
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