Identification and Characterization of a Single High-Affinity Fatty Acid Binding Site in Human Serum Albumin

Article abstract of DOI:10.1002/anie.201710437

A single high-affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site-specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA-binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow-sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X-ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug-protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.

Full text of DOI:10.1002/anie.201710437

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Accepted Article
Title: Identification and Characterization of a Single High-Affinity Fatty
Acid Binding Site in Human Serum Albumin
Authors: Stefan Petry, Lea Wenskowsky, Herman Schreuder, Volker
Derdau, Hans Matter, Julia Volkmar, and Till Opatz
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201710437
Angew. Chem. 10.1002/ange.201710437
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Identification and Characterization of a Single High-Affinity Fatty
Acid Binding Site in Human Serum Albumin
Lea Wenskowsky,[b] Herman Schreuder,[a] Volker Derdau,[a] Hans Matter,[a] Julia Volkmar,[c] Till
Opatz,[b]* and Stefan Petry[a]*
Abstract: A single high-affinity fatty acid binding site in the important
human transport protein serum albumin (HSA) is identified and
characterized using a NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-C12
fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA
complex with high site-specificity. The complex dissociation constant
is determined by titration experiments as well as radioactive
equilibrium dialysis. Competition experiments with known HSA-
binding drugs warfarin and ibuprofen confirm the new binding site to
be different from Sudlow-sites I and II. These binding studies are
extended to other albumin binders and fatty acid derivatives.
Furthermore an X-ray crystal structure allows locating the binding
site in HSA subdomain IIA. The knowledge about this novel HSA site
will be important for drug depot development and for understanding
drug-protein interaction, which are important prerequisites for
modulation of drug pharmacokinetics.
anions with delocalized charge, site II is specific for aromatic
carboxylates. Based on their preference for two important drug
molecules, these sites were also named “warfarin site” and
“ibuprofen site”.[5a] He and Carter located these binding sites in
the subdomains IIA (Sudlow-site I) and IIIA (Sudlow-site II) of
HSA’s structure (Figure 1) by X-ray crystallography.[6]
Investigations of the interaction between HSA with fatty acids
were reported by various groups.[7] For example, Curry et al.
postulated up to seven distinct fatty acid binding sites in HSA
from X-ray crystallography, as indicated by numbers in Figure
1.[8]
The detailed understanding of ligand interactions with the
abundant human transport protein serum albumin (HSA) is of utmost
importance for today’s drug discovery, since many common drugs
are known to interact with HSA. HSA binding has outstanding effects
on drug transport, release and solubility, decreases aggregate
formation and overall usually prolongs the half-life of a drug. It
directly impacts drug-repository effects as well as drug-drug-
interactions through competition of multiple ligands for the same
binding site.[1] Blood contains a significant amount, about 35–52 g/L,
of this 66 kDa protein,[2] which is synthesized by the liver at a rate of
~0.2 g/day/kg body weight.[3] HSA has important physiological
functions, including maintenance of colloid osmotic pressure and pH
value.[2] Furthermore, it serves as carrier and depot for small
lipophilic molecules such as fatty acids, bile acids, lipids, bilirubin,
steroid hormones and various nonpolar drug molecules.[2,4] Due to
potential competition for specific binding sites on HSA, the
interactions of drugs with dietary lipids, in particular free fatty acids,
are worth to be investigated.
Figure 1. Schematic view of the X-ray crystal structure of a 7:1 complex of
myristic acid and HSA (PDB 1e7g, resolution: 2.50 Å)[8] with Sudlow binding
sites[6]
.
Not all HSA binding sites display the same affinity to fatty
acid ligands. Different experimental approaches have been used
to determine dissociation constants KD of the representative fatty
acid myristic acid (C14, 5), including partition dialysis, equilibrium
dialysis, isothermal calorimetry and high-performance affinity
chromatography.[7e,9] Given the physiological function of HSA to
bind and deliver lipophilic metabolites through the bloodstream,
the low experimental KD values reported (30 to 250 nM)[7e,9-10]
are somewhat unexpected.
Pioneering work on interactions of HSA with drugs was
reported by Sudlow et al. in the 1970’s and two distinct binding
sites, Sudlow-sites I and II, have been identified by competition
experiments.[5] While binding site I is specific for heterocyclic
The goal of our study is to investigate high affinity binding
sites for fatty acids in HSA in detail. Considering the high
conformational flexibility of HSA,[2] fluorescence spectroscopy,
equilibrium dialysis and X-ray crystallography might be suitable
methods. First, we report a new fluorescent label without
intrinsic affinity to HSA. This label is linked to fatty acids of
different chain lengths to demonstrate binding and stoichiometry
of the resulting conjugates to HSA. From competition
experiments of the best label-fatty acid conjugate with unlabeled
myristic acid (5), we show that a carboxylate moiety in this
ligand is essential for specific binding. The site is further
[a]
[b]
[c]
Dr. H. Schreuder, Dr. V. Derdau, Dr. H. Matter, Dr. S. Petry
Sanofi-Aventis Deutschland GmbH, R&D, IDD
Industriepark Höchst, 65926 Frankfurt am Main, Germany
E-mail: StefanMatthias.Petry@sanofi.com
L. Wenskowsky, Prof. Dr. T. Opatz
Institute of Organic Chemistry, Johannes Gutenberg-University
Duesbergweg 10–14, 55128 Mainz, Germany
E-mail: opatz@uni-mainz.de
J. Volkmar current address
Provadis School of International Management and Technology
65926 Frankfurt am Main, Germany
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/anie.201710437
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COMMUNICATION
characterized by X-ray crystallography. Finally, we tested a
variety of ligands in fluorometric competition assays.
the importance of the carboxylate using a 25 µM concentration
of the 1:1 3f/HSA complex (Figure 2D).
For investigation of lipid-HSA interactions,
a polarity-
A
B
100
80
60
40
20
0
sensitive molecular probe is required. Dansyl (5-
(dimethylamino)naphthalene-1-sulfonyl), a fluorescent label, has
already been applied for binding and competition studies.[5b,11]
Sudlow et al. used dansylated substrates to characterize two
drug binding sites in albumin.[5b,11f] Dansylglycine and
dansylsarcosine revealed a 2:1 binding stoichiometry, as did –
even more surprisingly – dansylamide itself. This unmasks the
intrinsic affinity of the dansyl label, as noted by Curry et al.,[11g]
and is also consistent with our results (Figure S3). Thus, an
“innocent” fluorophore is needed to minimize nonspecific
interactions. The new fluorescence label should be small, devoid
of intrinsic HSA affinity, and exhibit a high quantum yield and a
large Stokes shift. NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)[12]
was the only label in our hands that fulfils these requirements.
Studies in lipid chemistry also show that NBD-labelled
substrates are readily accepted by acyltransferases, neutral
lipases, phospholipases and HSA.[13] Moreover, NBD is a “turn-
on” fluorophore, the quantum yield of which drastically increases
by adsorption onto the lipophilic surface of HSA.[14]
100
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(µ M
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)
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S A
Figure 2. A) Titration of HSA (0–200 µM) to NBD-labelled derivatives 3a (),
3b (), 3c (), 3d (), 3e (), 3f () (12.5 µM). B) Titration of NBD-labelled
derivative 3f (0–400 µM) to HSA (12.5 µM). C) Determination of the binding
stoichiometry of 3f to HSA (12.5 µM). A titration curve and a Job Plot for a
higher HSA concentration (25 µM; KD = 22 ± 3 µM) are given in the Figures
S5–6. D) Competition experiments of myristic acid (5;) and myristic acid
methyl ester (6;) against 3f on HSA (25 µM each).
-Aminocarboxylic acids 2a–f of different chain lengths were
reacted with NBD-Cl (1) to form NBD conjugates 3a–f,[15] which
in turn were titrated to HSA to detect changes in fluorescence
(Scheme 1).
While myristic acid (5) shows the expected competition
with 3f for HSA, resulting in a decrease of the fluorescence
signal, methyl ester 6 only competes at high concentrations
(Figure 5). Thus, a carboxylate function appears to be essential
for binding to the new HSA binding site, while binding is not
driven by lipophilicity alone. Based on these data, we expected
that the NBD-fatty acid binding site to contain at least one basic
amino acid side chain as potential partner in a salt-bridge with
the carboxylate of myristic acid (5) but not with its ester 6.
Overall, we conclude that NBD-FA 3f binds to albumin with a
new binding mode or to a new site. Our experiments have
shown that saturation is reached at a NBD-fatty acid/HSA-ratio
of approximately 4:1-6:1.
Scheme 1. Synthesis of NBD-fatty acid conjugates 3a–3f with increasing
chain lengths (C2–C12).
Binding of 3f to HSA was studied by crystallography (see
Supporting Information). The crystal obtained was anisotropic
and diffracted to 2.4 Å in the best direction, and to 3.1 Å in the
other two directions. The conformation of our HSA molecule is
more similar to the fat-free conformation (2.8 Å rmsd with the fat-
free 1uor structure[6]) than to the fatty-acid bound conformation
(5.3 Å rmsd with the myristate-bound 1e7g structure[8]). There is
clear electron density found for the largest part of 3f (Figure 3A).
Possible ligand conformations were refined, obtained by docking
using GlideXP[18] plus MM-GBSA[19] scoring with the OPLS3
force field[20] and flexible side chains 3 Å around the ligand. The
conformation of 3f in Figure 3a resulted in the best fit to the
electron density maps, revealed optimal interactions to HSA and
was also the best scoring pose.
While short chain conjugates 3a–3c (C2–C6) did not show
an increase in fluorescence upon addition to HSA, thus
suggesting the lack of an intrinsic affinity of the NBD label to
HSA, the more lipophilic derivatives 3d–3f (C8–C12) have the
desired “turn-on” characteristics, indicative of binding to a
lipophilic region of the protein (Figure 2A). The experimental
titration curve of the NBD-labelled lauric acid 3f shows the
expected increase in fluorescence with a well-defined saturation
behavior (Figure 2B). A curve fit[16] for saturation of a single
binding site in Figure 3 shows a high correlation coefficient (r2 =
0.96) and a KD of 27 ± 2 µM. This value was independently
confirmed by dialysis experiments with NBD-labelled lauric acid
3f (KD = 14 ± 6 µM, see Figure S3). Moreover, a Job plot[17] for
the fluorescence intensity in the 3f/HSA-system confirms the
assumed 1:1 binding stoichiometry (Figure 2C). Next,
competition experiments were performed with unlabeled myristic
acid (5) and its methyl ester 6 as competing ligand to investigate
As shown in Figure 3a, 3f (orange carbon atoms) is bound
to HSA subdomain IIA; its nitro group interacts with side chains
of Ser202 and Trp214, while the carboxylate group interacts with
the side chains of Lys199 and His242. The 4-NBD group is
stacked between the indole ring of Trp214 and the aliphatic part
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10.1002/anie.201710437
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COMMUNICATION
acceptor. The emission spectra of HSA show the expected
decrease with increasing amounts of 3f suggesting an energy
transfer between the Trp214 and the NBD group of 3f (Figure
4A). A titration of 3f to a 1:1 7/HSA complex still results in an
increase of the fluorescence with a well-defined saturation
behavior (KD = 24 ± 4 µM; Figure 4B). These data support the
assumed existence of neighboring cavities. To determine the
influence of the introduction of the NBD label on fatty acid
binding to HSA, dialysis experiments were performed using
tritium-labelled myristic acid (5). Figure 4C shows the
experimental data and the theoretical curve[16] reflecting a high
correlation coefficient (r2 = 0.99). The apparent KD of [3H]myristic
acid (5) assuming a 1:1 binding model was determined to be
0.94 ± 0.1 µM, which demonstrates that NBD-labelling reduces
affinity by about 20-fold but does not prevent specific binding.
A
B
120
80
40
0
150
100
50
[N B D -F A ]
Figure 3. A) X-ray crystal structure of 3f in complex with fatty acid free human
serum albumin (PDB 6ezq). 3f (orange carbon atoms) binds to HSA
subdomain IIA. Key amino acids and interactions are indicated (yellow: H-
bonds, cyan: - interactions). The ligand conformation revealed the best fit to
the electron density maps and is in agreement with docking (GlideXP). B)
Binding site analysis of the HSA-3f complex using SiteMap. Red regions
indicate favourable potential H-bond acceptor interactions; blue regions
indicate favourable H-bond donor interactions and yellow regions indicate
favourable hydrophobic interactions. C) WaterMap hydration sites from 10 ns
MD simulation mapped onto the binding mode of 3f. Only sites overlapping
with 3f with a fractional overlap of 0.1 to 1 are shown to illustrate energetically
unfavourable and favourable sites. Green spheres indicate stable hydration
sites with more negative G values; red-brown spheres indicate more positive
G values, i.e. more instable sites. (D) Postulated binding mode of
sulfasalazine (10; green carbon atoms) in the same HSA site as 3f (GlideXP
and MM-GBSA). Nonpolar hydrogen atoms are omitted for clarity
0
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(n m
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Figure 4. A) Emission spectra of HSA (25 µM) with increasing amounts of 3f
(0–400 µM). B) Titration of NBD-labelled derivative 3f (0–400 µM) to 7/HSA
(25 µM). ) C) Amount of [3H]myristic acid (5;12.5 µM) bound to HSA (0.05–25
µM). D) Competition experiment of sulfasalazine (10) against 7 on HSA (25
µM).
of the Lys199 side chain. This latter region is also occupied by
other aromatic HSA ligands[21] in PDB.[22] We detected additional
electron density in Sudlow site II representing a partially bound
second molecule 3f (see Supporting Information).
The main binding site was analyzed by SiteMap[23] to
identify favorable interactions, which are in agreement to the key
interactions outlined above. Furthermore, an analysis of
hydration sites was performed using WaterMap[24] from a 10 ns
MD simulation without ligand (see Supporting Information). For
visualization, only hydration sites overlapping with 3f are
displayed. In fact, the ligand replaces energetically unfavorable
(brown-red) hydration sites next to its aromatic ring, the nitro
group and the aliphatic linker. More favorable (green) hydration
sites are located close to the ligand carboxylate, which is caused
by desolvation penalties around polar residues in this region.
WaterMap employs a statistical thermodynamics analysis of
hydration sites following MD. This allows estimating entropy,
enthalpy and free energy of water molecules around ligands in
binding sites. Energetic values are compared to bulk reference
values to estimate the enthalpic cost and excess entropy to
transfer water from a hydration site to bulk solvent, which is our
basis for classification into favorable and unfavorable sites.
FRET[25] experiments then confirmed the -stacking
between Trp214 (donor) and the 4-NBD group (acceptor). Figure
S7 exhibits the required spectral overlap between donor and
We finally aimed to identify more ligands capable of
competing with 3f for HSA binding. To this end, a series of drugs
and other compounds, including known HSA-ligands (Figure 5a,
S9, Table S2), have been employed for competition experiments
using a 25 µM concentration of the 1:1 3f/HSA complex. While
ibuprofen (8) shows no appreciable effect at all concentrations,
warfarin (7) leads to a steep increase of fluorescence intensity.
Clearly, these drugs are not competitive to 3f, as this would
result in decreased fluorescence, as seen for myristic acid (5). In
contrast, no influence is expected for a ligand occupying a
different ligand binding site in HSA and not affecting the protein
conformation. This apparently is the case for ibuprofen (8),
binding in subdomain IIIA.[6]
The increase of fluorescence intensity for warfarin (7) is
surprising at first glance but can be explained by a change of
HSA’s conformation upon binding of 7, which in turn alters the
geometry of the 3f binding site. This “paradox” behavior of
warfarin (7) and other ligands binding to Sudlow-site I (warfarin
site) has previously been reported.[5b]
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COMMUNICATION
In summary, we have characterized a binding site for fatty
acids on human serum albumin using a NBD-labelled C12 fatty
acid (3f) as a specific fluorescent ligand. Ligand 3f shows a 1:1
binding stoichiometry in a complex with HSA. The introduction of
the NBD-NH-moiety reduces the affinity but does not alter
binding. Myristic acid (5) as well as sulfasalazine (10) and
balsalazide (11) were found to be competing ligands.
It can be assumed, that many other drugs bind into this
high affinity fatty acid binding site.
This novel probe will allow a better understanding of drug
protein binding and enable new applications in drug depot
development and modulation of pharmacokinetics. Competition
experiments using a combination of ligands specific for different
binding sites will permit detailed insights into binding of endo-
and exogenous ligands to human serum albumin and allow the
analysis of drug-drug and food-drug interactions. Another aspect
is the investigation of the effect of states of disease on albumin
binding as the plasma levels of free fatty acids may alter the
drug binding capacity of HSA through the newly described
binding site. Currently, we are extending our studies on various
other competing drugs and to other lipids at higher but still
physiologically relevant concentrations as well as on other assay
systems.
Figure 5. A) Structures of tested albumin binders. B) Competition experiments
of warfarin (7;) and ibuprofen (8;) against 3f on HSA (25 µM each). C)
Competition experiments of 3-(decyloxy)benzoic acid (9;), sulfasalazine
(10;) and bumetanide (12;) against 3f on HSA (25 µM each).
Experimental Section
For experimental details, crystallographic data and further biochemical
data, see the Supporting Information.
In search for other ligands, we found balsalazide (11) and
sulfasalazine[26] (10), which has been proposed to bind to HSA
subdomain IIA based on fluorescence quenching experiments[27]
to act as competitive binders in the NBD-fatty acid binding site.
The postulated binding mode for 10 from GlideXP[18] docking
followed by MM-GBSA[19] scoring using the HSA-3f X-ray
structure is displayed in Figure 3D (green carbon atoms); here
the salicylate is possibly involved in the - interaction to Trp214,
while the position of the distal pyridyl is less clearly defined in
high-ranked poses. In a competition experiment of sulfasalazine
(10) against warfarin (7) as label for drug site 1[5a] a decreasing
fluorescence signal can be observed (Figure 4D) meaning
sulfasalazine (10) competes with 3f and 7 at comparable
concentrations.
In contrast, the arene-containing lipophilic acid 3-
(decyloxy)benzoic acid (9) showed no effect on fluorescence
intensity. In addition to warfarine (7), bumetanide (12) is another
drug increasing the fluorescence signal during the titration to
HSA and can thus be grouped in the same class with warfarin.[28]
Therefore we conclude that the binding site for the NBD-
fatty acid conjugate 3f is not identical with the ibuprofen or
warfarin site in HSA but partly overlap with the latter, which
could explain the increase in the fluorescence of bound 3f upon
filling of the warfarin site. As warfarin (7) does not displace 3f
from HSA, a shared occupancy in neighboring subsites in this
region can be assumed. As demonstrated for the myristate/9
pair, 3f permits the discrimination between lipophilic acids of
different shape through HSA.
,
Acknowledgements
We thank Dr. J. Liermann (Mainz) for NMR spectroscopy, Dr. N.
Hanold (Mainz, deceased) for mass spectrometry, Alexander
Liesum for help with crystallization and mounting of the crystal,
Joachim Diez for data collection and Petra Loenze for data
processing.
Keywords: Fatty Acid • Binding Site • Human Serum Albumin •
NBD Label • Drug Interaction
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10.1002/anie.201710437
Angewandte Chemie International Edition
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COMMUNICATION
It´s not a hydrophobic
sponge: The
identification and
Lea Wenskowsky, Herman
Schreuder, Volker Derdau,
Hans Matter, Julia
characterization of a
single high-affinity fatty
acid binding site in
human serum albumin
has been achieved using
a highly specific binding
NBD-labelled fatty acid.
An X-ray crystal structure
locates the binding site in
subdomain IIA. The
identified binding site
serves as a tool for
understanding of drug
protein binding,
Volkmar, Till Opatz*, and
Stefan Petry*
Page No. – Page No.
Identification and
Characterization of a
Single High Affinity Fatty
Acid Binding Site in
Human Serum Albumin
modulation of
pharmacokinetic profiles
and drug depot
development.
Author(s), Corresponding Author(s)*
((Insert TOC Graphic here))
Page No. – Page No.
Title
Text for Table of Contents
This article is protected by copyright. All rights reserved.