Báo cáo khoa học: Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs An optical and NMR spectroscopic study potx

Modulation of heme and myristate binding to human
serum albumin by anti-HIV drugs
An optical and NMR spectroscopic study
Gabriella Fanali
1
, Alessio Bocedi
2,3,
*, Paolo Ascenzi
2,3
and Mauro Fasano
1
1 Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Universita
`
dell’Insubria, Busto Arsizio, Italy
2 Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Rome, Italy
3 Dipartimento di Biologia, and Laboratorio Interdisciplinare di Microscopia Elettronica, Universita
`
‘Roma Tre’, Rome, Italy
Human serum albumin (HSA) is the most prominent
protein in plasma (its concentration being 45 mgÆmL
)1
,
i.e. 7.0 · 10
)4
m, in the serum of adults), but it is also
found in tissues and secretions throughout the body
[1]. HSA is made up of a single nonglycosylated
all-a chain of 65 kDa, containing three homologous
domains (I, II and III). Each domain is made up of
two separate helical subdomains (A and B), connected
by random coils (Fig. 1) [1–7]. The HSA globular

domain structural organization provides a variety of
binding sites for various ligands, making it an impor-
tant determinant of the pharmacokinetic behaviour of
many drugs [1,3,5–12]. Moreover, it accounts for most
of the antioxidant capacity of human serum, acts as a
nitric oxide depot and displays enzymatic properties
[1,5,13–18].
Among other ligands, HSA is able to bind up to seven
equivalents of long chain fatty acids (FAs) at multiple
binding sites (labelled FA1–FA7; Fig. 1) with different
affinity [5,12,19–22]. Remarkably, FA7 represents
Keywords
allosteric modulation; anti-HIV drugs;
heterotropic interactions; human serum
albumin; NMR relaxation
Correspondence
M. Fasano, Dipartimento di Biologia
Strutturale e Funzionale, and Centro di
Neuroscienze, Universita
`
dell’Insubria,
Via Alberto da Giussano 12, I-21052 Busto
Arsizio (VA), Italy
Fax: +39 0331 339459
Tel: +39 0331 339450
E-mail:
*Present address
Istituto di Ricerche di Biologia Molecolare
‘P. Angeletti’, Rome, Italy
(Received 27 February 2007, revised

20 June 2007, accepted 5 July 2007)
doi:10.1111/j.1742-4658.2007.05978.x
Human serum albumin (HSA) has an extraordinary ligand-binding capac-
ity, and transports Fe(III)heme and medium- and long-chain fatty acids. In
human immunodeficiency virus-infected patients the administered drugs
bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)-
heme to HSA in the presence of three representative anti-HIV drugs and
myristate is investigated. Values of the dissociation equilibrium constant K
d
for Fe(III)heme binding to HSA were determined at different myristate
concentrations, in the absence and presence of anti-HIV drugs. Nuclear
magnetic relaxation dispersion profiles of HSA–Fe(III)heme were mea-
sured, at different myristate concentrations, in the absence and presence of
anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are
provided by automatic docking simulation. Abacavir and nevirapine bind
to HSA with K
d
values of 1 · 10
)6
and 2 · 10
)6
m, respectively. Therefore,
at concentrations used in therapy (in the 1–5 · 10
)6
m range) abacavir and
nevirapine bind to HSA and increase the affinity of heme for HSA. In the
presence of abacavir or nevirapine, the affinity is not lowered by myristate.
FA7 should therefore be intended as a secondary binding site for abacavir
and nevirapine. Binding of atazanavir is limited by the large size of the
drug, although preferential binding may be envisaged to a site positively

coupled with FA1 and FA2, and negatively coupled to FA7. As a whole,
these results provide a foundation for the comprehension of the complex
network of links modulating HSA-binding properties.
Abbreviations
FA, fatty acid; HSA, human serum albumin; NMRD, nuclear magnetic relaxation dispersion.
FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS 4491
Sudlow’s site I, the preferential binding site for bulky,
heterocyclic anions (e.g. warfarin), whereas the cavity
hosting FA3 and FA4 contributes to Sudlow’s site II,
which is preferred by aromatic carboxylates with
an extended conformation (e.g. ibuprofen), benzodia-
zepines (e.g. diazepam) and some anaesthetics
[1,5,7,8,11,23–26].
FA1, located in subdomain IB (Fig. 1), hosts the
heme, with the tetrapyrrole ring arranged in a
D-shaped cavity limited by Tyr138 and Tyr161 resi-
dues, which provide p–p stacking interaction with the
porphyrin and supply a donor oxygen (from Tyr161)
for the Fe(III)heme iron [27,28]. Interestingly, FA1 has
a low affinity for long- and medium-chain FAs, sug-
gesting that its structure has evolved to specifically
bind the heme [21–29].
HSA undergoes pH- and allosteric effector-
dependent reversible conformational isomerization(s).
Between pH 4.3 and 8.0, in the absence of allosteric
effectors, HSA displays the neutral (N) form that is
characterized by a heart-shaped structure (Fig. 1). At
pH values > 8.0, in the absence of allosteric effectors,
HSA changes conformation to the basic (B) form
(neutral-to-basic, N fi B transition) with loss of the

a -helix content and an increased affinity for some
ligands [1,6,7,30–37]. Ligand binding to HSA stabilizes
protein conformers N or B, thus regulating allosterical-
ly the conformational transition(s). Heme regulates
drug binding to Sudlow’s site I by heterotropic inter-
actions. Indeed, the affinity of Fe(III)heme for HSA
decreases by about one order of magnitude upon drug
binding, and accordingly Fe(III)heme binding to HSA
decreases drug affinity by the same extent. Therefore,
drugs that bind to Sudlow’s site I (e.g. warfarin) act as
allosteric effectors for Fe(III)heme association, and
vice versa. Also benzodiazepines bind to several
functionally and allosterically linked HSA clefts,
depending on their optical conformation and substitu-
tion [35,38–42].
In HIV-infected individuals the primary target of
therapy is the HIV itself, but most of the clinical mani-
festations are related to the effect of HIV on the
immune system, which leads to progressive immunode-
ficiency. Recently, the introduction of highly effective
combination regimens of antiretroviral drugs has led
to substantial improvements in morbidity and mortal-
ity [43]. The anti-HIV drugs include three different
classes among nucleoside reverse transcriptase inhibi-
tors, non-nucleoside reverse transcriptase inhibitors
and protease inhibitors. Nucleoside reverse transcrip-
tase inhibitors are intracellularly phosphorylated
to their corresponding triphosphorylated derivatives,
which compete with the corresponding natural nucleo-
tide for binding to HIV reverse transcriptase and inhi-

bit it. Non-nucleoside reverse transcriptase inhibitors
act as noncompetitive inhibitors of the HIV reverse
transcriptase. Protease inhibitors interfere with viral
replication by inhibiting the viral protease, preventing
maturation of the HIV virus and causing the forma-
tion of noninfectious virions [43–48]. The therapeutic
efficiency of anti-HIV drugs in combination therapy is
strictly dependent upon the mutual interaction(s) of
binding equilibria with plasma proteins and in particu-
lar with HSA. One of the most important factors
affecting the distribution and the free, active concen-
tration of many administered drugs is binding affinity
for HSA [7,49–51].
Here, the effect of myristate on the binding of
Fe(III)heme to HSA in the absence and presence of
three anti-HIV drugs belonging to different pharmaco-
logical classes, i.e. abacavir, nevirapine and atazanavir
(Scheme 1), is reported by means of optical and mag-
netic spectroscopy. Moreover, a screening of potential
HSA-binding sites has been performed by automated
docking simulation for the different anti-HIV drugs
considered.
Results
Fe(III)heme was titrated with HSA by measuring the
difference in absorbance at 411 nm in the UV–Vis
spectrum (DA; see Eqn 1) with respect to the spectrum
Fig. 1. HSA structure. Atomic coordinates were taken from PDB
entry 1O9X [28]. FA-binding sites are indicated by arrows and
labelled. Site FA1 is occupied by heme (red). Sites FA2–FA7 are
occupied by myristate (green). FA7 represents Sudlow’s site I (i.e.

the warfarin site). FA3 and FA4 together represent Sudlow’s site II
(i.e. the ibuprofen site). For further details see text.
Modulation of HSA ligand binding by anti-HIV drugs G. Fanali et al.
4492 FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS
in the absence of HSA at different myristate concen-
trations (Fig. 2) in the absence (Fig. 2A) and presence
of abacavir (Fig. 2B), nevirapine (Fig. 2C) and ataz-
anavir (Fig. 2D), respectively. In agreement with a
similar experiment performed on Mn(III)heme [36], in
the absence of any drug (Fig. 2A) myristate may
compete for the heme site with a twofold reduction in
the affinity of heme for HSA (Table 1). In the presence
of abacavir (Fig. 2B), positive cooperation is observed
in the absence of myristate: therefore, the K
d
value
cannot be obtained using Eqn (1). In the presence of
myristate the curves assume a hyperbolic behaviour
and the values reported in Table 1 are obtained. The
affinity of heme for HSA is slightly improved even in
the presence of myristate. Binding isotherms obtained
in the presence of nevirapine (Fig. 2C) tend to an
asymptotic DA
max
value higher than that observed in
the absence of any drug and in the presence of abaca-
vir. In the absence of myristate and at a myristate con-
centration of 1.0 · 10
)5
m, curves cannot be fitted

using Eqn (1): however, at higher myristate concentra-
tions the K
d
values are consistent with an increased
affinity of HSA for heme. In the presence of atazana-
vir (Fig. 2D), a hyperbolic binding isotherm is
observed, affording the determination of K
d
¼
7.4 · 10
)8
m, a value definitely smaller than those
observed in the absence or presence of other allosteric
effectors. When myristate is added, a more compli-
cated situation occurs with binding isotherms that
show features observed for both other drugs as well.
The asymptotic DA
max
value changes with the myri-
state concentration, the value in the absence of myri-
state being lower than the corresponding value in the
absence of any drug.
In order to check the possibility that abacavir, nevi-
rapine and atazanavir could fit into the heme (FA1)
binding site, as well as into Sudlow’s site I (FA7), or
into the other two sites in close proximity (FA2 and
FA6), an automated docking analysis of the three
drugs was performed in the four binding sites. Inter-
molecular energy values are reported in Table 2. Fig-
ure 3 shows a ribbon model of the HSA FA1 region

with abacavir and nevirapine superimposed on Fe(III)-
heme, whereas Figs 4–6 show ribbon models of abaca-
vir, nevirapine and atazanavir superimposed on
myristate in sites FA2, FA6 and FA7, respectively. As
can be seen, abacavir (Figs 3A, 5A, and 6A) fits
reasonably well in sites FA1, FA6 and FA7, whereas
docking to site FA2 is disadvantaged. Nevirapine
(Figs 3B, 4, 5B, and 6B) is able to enter all four sites,
thus competing with their ligands and potentially
acting as an allosteric effector. By contrast, atazanavir
(Fig. 5C) could partially enter the binding cavities,
although steric clashes between this large ligand and
the protein matrix make it unlikely, except for the
extended FA6 trough.
Paramagnetic Fe(III)heme–HSA(–drug) complexes
were also investigated in terms of their ability to relax
solvent water protons at different proton Larmor fre-
quencies. When the magnetic field is rapidly changed
from a low to a high value, the magnetization expo-
nentially changes to reach its equilibrium value with a
time constant that is T
1
in the new magnetic field.
Therefore, measurement of magnetization at progres-
sive time intervals allows us to obtain the T
1
value. In
order to read the magnetization value, it has to be
transformed into an electromagnetic signal by a radio-
frequency pulse at its Larmor frequency that depends

linearly from the magnetic field. Therefore, the field
needs to be re-switched at a unique value correspond-
ing to the frequency at which the transmitter and the
receiver are tuned. Such a fast change of the magnetic
field between equilibration, evolution (relaxation) and
detection values is called fast-field cycling. If this
Scheme 1.
G. Fanali et al. Modulation of HSA ligand binding by anti-HIV drugs
FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS 4493
experiment is repeated for different magnetic fields (i.e.
at different proton Larmor frequencies) a nuclear
magnetic relaxation dispersion (NMRD) profile
is obtained. Figure 7 reports the NMRD profiles of
1.0 · 10
)4
m Fe(III)heme–HSA at different myristate
concentrations in the absence (Fig. 7A) and presence
of abacavir (Fig. 7B), nevirapine (Fig. 7C) and ataz-
anavir (Fig. 7D). By increasing the myristate concen-
tration, a slight smoothing of the curves is observed
in all cases. In the presence of abacavir (Fig. 7B), no
significant differences might be appreciated in compari-
son with Fig. 7A (i.e. in the absence of the anti-HIV
drugs). Remarkably, in the presence of nevirapine
(Fig. 7C) significant quenching of the low-frequency
Fig. 2. Binding isotherms for Fe(III)heme binding to HSA (FA free) and to HSA–myristate complexes in the absence of drugs (A) and pres-
ence of abacavir (B), nevirapine (C) and atazanavir (D), at pH 7.0 and 25 °C. In all panels, the binding isotherm measured in the absence of
either drugs or myristate is shown for comparison (solid squares); solid diamonds, no myristate; open upward triangles, 1.0 · 10
)5
M myri-

state; open downward triangles, 7.5 · 10
)5
M myristate; open diamonds, 1.0 · 10
)4
M myristate. The continuous lines were obtained by
analysis of the data by using Eqn (1), when applicable. For further details see text.
Table 1. Values of the thermodynamic dissociation constants (K
d
, M) for Fe(III)heme–HSA in the absence and presence of abacavir, nevira-
pine and atazanavir, at different myristate concentration, pH 7.0 and 25 °C.
[Myristate] (
M) No drug Abacavir Nevirapine Atazanavir
0 (5.0 ± 0.2) · 10
)7a a
(7.4 ± 3.0) · 10
)8
1.0 · 10
)5
(5.3 ± 0.4) · 10
)7
(2.9 ± 0.4) · 10
)7a a
7.5 · 10
)5
(1.3 ± 0.2) · 10
)6
(2.5 ± 0.4) · 10
)7
(2.9 ± 0.3) · 10
)7a

1.0 · 10
)4
(1.3 ± 0.1) · 10
)6
(5.1 ± 0.8) · 10
)7
(1.1 ± 0.2) · 10
)7a
a
The binding isotherm deviates significantly from the hyperbolic behaviour, therefore data cannot be analysed in terms of Eqn (1).
Modulation of HSA ligand binding by anti-HIV drugs G. Fanali et al.
4494 FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS
region is observed that is further emphasized by the
presence of myristate. Atazanavir (Fig. 7D) does not
affect the NMRD profile of Fe(III)heme–HSA in the
absence of myristate, although a moderate decrease at
low frequency and a remarkable smoothing might
be appreciated. Noticeably, an exhaustive theoretical
treatment of the NMRD profiles of high-spin Fe(III)-
heme protein complexes would require extensive com-
putational work and is beyond the scope of this study.
Discussion
Allosteric modulation of HSA-binding properties is
fundamental for a safe management of patients subject
to multidrug therapy, affecting the distribution and the
free concentration of each administered drug. While a
certain extent of HSA interaction may be desirable to
help drug solubilization and distribution, a too tight
an interaction negatively affects the distribution to
sites of action and dramatically increases the total con-

centration of the administered drug [7,35,50,51]. How-
ever, negative modulation of the drug–HSA affinity by
heterotropic interactions would suddenly increase the
drug concentration which may reach the toxicity
threshold [7,31,35,36,51–55]. Here, we show how drugs
currently used at the micromolar level in highly active
antiretroviral therapy may bind to different sites thus
causing opposite effects on the conformational states
of HSA. Myristate, binding to all FA sites, competes
with Fe(III)heme in FA1 determining an increase of
K
d
by a factor of two and at the same time modulates
the N fi B transition and stabilizes the binding of
Fe(III)heme. This finding is in agreement with a simi-
lar behaviour observed for the less-stable Mn(III)-
heme–HSA complex [36].
Abacavir has been recently reported to be a Sud-
low’s site I (FA7) ligand because of its ability
to quench Trp214 fluorescence and the negative
modulation it exerts on heme binding [51]. Indeed, a
thorough analysis of several HSA-binding drugs has
been reported showing that all FA7 ligands reduce the
affinity of heme by one order of magnitude and,
accordingly, heme reduces by one order of magnitude
the affinity of all FA7 ligands [35]. However, the
results shown here indicate that abacavir binding
involves multiple sites and FA7 is probably not the
primary binding site for abacavir. Indeed, abacavir
A

B
Fig. 3. Superimposition of Fe(III)heme and abacavir (A) and nevira-
pine (B) in binding site FA1. Ligands are coloured as follows: heme,
red; abacavir, blue; nevirapine, orange. Atomic coordinates were
taken from the PDB entry 1O9X [28]. For further details see text.
Table 2. Values of intermolecular energies (kJÆmol
)1
) obtained from
the docking simulation of the three anti-HIV drugs in the heme
binding cavity (FA1) and in the functionally linked binding sites. ND,
not determined.
Abacavir Nevirapine Atazanavir
FA1 ) 30.6 ) 26.5 $ 10
3
FA2 ND ) 20.3 $ 10
4
FA6 ) 28.9 ) 30.7 ) 11.2
FA7 ) 31.6 ) 33.5 $ 10
3
G. Fanali et al. Modulation of HSA ligand binding by anti-HIV drugs
FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS 4495
binds to HSA at K
d
¼ 10
)6
m (Supplementary mate-
rial), two orders of magnitude lower than that
obtained by fluorescence quenching [51]. On the basis
of the docking simulations, abacavir would also bind
to FA1, competitively preventing the binding of heme.

At concentrations used previously [51], abacavir may
enter the FA7 cavity and block the conformational
switch towards the B form, characterized by an
increased affinity for FA1 ligands (Supplementary
material). The actual ability of abacavir to fit FA1,
FA6 and FA7 is explored by docking simulations. In
the absence of experimental 3D structures of
HSA–anti-HIV complexes, docking procedures based
on Monte Carlo-simulated annealing are effective tools
for the screening of binding possibilities for the differ-
ent drug–HSA interactions [56–58]. As shown in
Fig. 3A, abacavir fits FA1, competing with heme;
abacavir may also fit into FA6 (Fig. 5A); conversely,
Fig. 6A shows that abacavir may also enter FA7 and
consequently lower the affinity for FA1, FA1 and FA7
being functionally linked [21,31,35,51]. Although the
structural basis for the observed allosteric regulation
of heme binding by Sudlow’s site I ligands is not
known, it has been suggested that it may be mediated
by rearrangement of the Phe149–Tyr150 dyad, Phe149
A
B
C
Fig. 4. Superimposition of myristate and nevirapine in binding site
FA2. Ligands are coloured as follows: myristate, green; nevirapine,
orange. Atomic coordinates were taken from the PDB entry 1O9X
[28]. For further details see text.
Fig. 5. Superimposition of myristate and abacavir (A), nevirapine (B)
and atazanavir (C) in binding site FA6. Ligands are coloured as fol-
lows: myristate, green; abacavir, blue; nevirapine, orange; atazana-

vir, cyan. Atomic coordinates were taken from the PDB entry 1O9X
[28]. For further details see text.
Modulation of HSA ligand binding by anti-HIV drugs G. Fanali et al.
4496 FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS
contacting the heme ring and Tyr150 protruding into
Sudlow’s site I (i.e. FA7) [21].
Nevirapine, a small hydrophobic butterfly-shaped
ligand, was reported to be a FA7 ligand as well, thus
acting as an allosteric negative effector of FA1. Also
in this case, this is just one of multiple binding modes
of nevirapine to HSA previously distinguished on the
basis of fluorescence quenching of Trp214 (located in
close proximity of FA7) [51]. Indeed, nevirapine binds
to its primary binding site at K
d
$ 2 · 10
)6
m (Supple-
mentary material). By looking at Fe(III)heme binding
it becomes clear that nevirapine acts as an allosteric
effector that increases the affinity of Fe(III)heme for
FA1. This positive modulation may be due to FA2
(Fig. 4), the only FA-binding site that contacts both
HSA domains I and II. A structural explanation would
involve Tyr150 again; actually, binding of myristate to
FA2 attracts Tyr150 and Arg252 towards the FA car-
boxylate moiety [21]. Therefore, Arg252 is no longer
available to stabilize FA7 ligands; however, the reori-
entation of Tyr150 may stabilize the interaction
of Phe149 with Fe(III)heme, thus explaining the allo-

steric modulation observed in solution studies
[7,18,31,35,36,39,51].
Atazanavir is a large, extended peptidomimetic
drug, and may fit multiple sites by partially entering
them. Although the four sites considered are poten-
tially able to host a more or less extended part of the
atazanavir molecule, the drug experiences steric hin-
drance due to residues that point outside the protein
core (Table 2). The only docking that shows stabiliza-
tion of the interaction energy takes place in FA6 for
its extended, open-trough conformation (Fig. 5C). As
a consequence, a situation occurs that is intermediate
between those observed for abacavir and nevirapine.
Interestingly, in the absence of myristate, ataza-
navir displays the larger stabilization effect on heme
affinity.
All these considerations are supported by the
NMRD data. Indeed, NMRD profiles recorded in the
absence (Fig. 7A) and presence (Fig. 7B) of abacavir
do not differ significantly; also, no differences are
observed by increasing the myristate-to-HSA ratio.
Nevirapine dramatically affects the profile by reducing
the R
1p
values at the low-frequency limit even in the
absence of myristate, the high-frequency region being
unaffected. Although extensive theoretical treatment of
the NMRD data is beyond the scope of this study, it
should be noted that such a smoothing of the NMRD
profile in high-spin Fe(III) complexes is usually

reported to be associated with a distortion of the zero-
field splitting tensor [60,61]. Indeed, in slowly rotating
systems the Solomon–Bloembergen–Morgan equation
breaks down and R
1p
values are affected by a number
of parameters arising from both contact and dipolar
electron–nucleus interactions, including anisotropies of
the g tensor, the zero-field splitting tensor, and the
hyperfine coupling tensor [62–64]. Interestingly, bind-
ing of nevirapine to HSA determines a remarkable dis-
tortion of the heme environment that reflects on both
NMRD profiles (Fig. 7C) and the asymptotic value of
the binding isotherms (Fig. 2C).
In conclusion, our results demonstrate that anti-HIV
drugs at concentrations used in highly active antiretro-
A
B
Fig. 6. Superimposition of myristate and abacavir (A) and nevirapine
(B) in binding site FA7. Ligands are coloured as follows: myristate,
green; abacavir, blue; nevirapine, orange. Atomic coordinates were
taken from the PDB entry 1O9X [28]. For further details see text.
G. Fanali et al. Modulation of HSA ligand binding by anti-HIV drugs
FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS 4497
viral therapy may allosterically exert heterotropic inter-
actions that influence reciprocally the Fe(III)heme- and
FA-binding properties to HSA. FA7, i.e. Sudlow’s
site I, is confirmed to be negatively linked to FA1, as
already established by solution studies [7,12,18,
21,31,35,36,39,51]. Thus, the increase in plasma levels

of Fe(III)heme under pathological conditions (e.g.
severe haemolytic anaemia, crash syndrome and post-
ischaemic reperfusion) may induce the release of FA7-
bound drugs with the concomitant intoxication of the
patient [7,35,51]. Moreover, binding of drugs to FA2
reduces the affinity of FA7 and increases the affinity
of FA1 ligands. Eventually, FA6 ligands are expected
to affect in some way the occupancy of FA1, as
evinced previously [21,36]. Thus, allosteric regulation
of ligand binding is relevant in pharmacological ther-
apy management, the nonspecific binding of drugs to
plasma proteins being an important determinant of
their biological efficacy by modulation of drug avail-
ability to the intended target.
Experimental procedures
Abacavir (GlaxoSmithKline, London, UK), nevirapine
(Boehringer Ingelheim, Ridgefield, CO), and atazanavir
(Bristol-Myers Squibb, Princeton, NJ) were obtained
through the NIH AIDS Research Reagent Program, Division
of AIDS, NIAID, National Institute of Health (Bethesda,
MD). All other reagents (from Sigma-Aldrich, St Louis,
MO), were of the highest purity available, and were used
without further purification. HSA (Sigma-Aldrich) was
essentially FA-free according to the charcoal delipidation
protocol [65–67] and was used without further purification.
Absence of significant amounts of covalent dimers was
checked using a Bruker Ultraflex MALDI-TOF mass spec-
trometer (Bruker Daltonics, Bremen, Germany).
Fig. 7. NMRD profiles of Fe(III)heme–HSA (FA free) and Fe(III)heme–HSA–myristate complexes in the absence of drugs (A) and in the presence
of abacavir (B), nevirapine (C) and atazanavir (D), at pH 7.0 and 25 °C. In all panels, the NMRD profile measured in the absence of either drugs or

myristate is shown for comparison (solid squares); solid diamonds, no myristate; open upward triangles, 1.0 · 10
)4
M myristate; open
diamonds: 4.0 · 10
)4
M myristate. Fe(III)heme–HSA concentration was 1.0 · 10
)4
M. R
1p
values were normalized to 1.0 · 10
)3
M. For further
details see text.
Modulation of HSA ligand binding by anti-HIV drugs G. Fanali et al.
4498 FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS
Fe(III)heme–HSA was prepared by adding the appropri-
ate volume of 1.2 · 10
)2
m Fe(III)heme, dissolved in
1.0 · 10
)1
m NaOH, to a 1.0 · 10
)4
m HSA solution in
0.1 m phosphate buffer pH 7.0. In the final HSA solution
Fe(III)heme–HSA was 1.0 · 10
)4
m.
The actual concentration of the Fe(III)heme stock solu-
tion was checked as a bis-imidazolate complex in SDS

micelles with an extinction coefficient of 14.5 mm
)1
Æcm
)1
(at 535 nm) [68]. Under all the experimental conditions, no
free Fe(III)heme was present in the reaction mixtures. The
actual concentration of the HSA stock solution was deter-
mined using the Bradford method [69].
Sodium myristate solution (0.1 m) was prepared by add-
ing 0.1 m FA to NaOH 1.0 · 10
)1
m. The solution was
heated to 100 °C and stirred to dissolve the FA. The
sodium myristate solution was cooled and then mixed with
1.0 · 10
)4
m Fe(III)heme–HSA (FA-free) to achieve the
desired FA to protein molar ratio. The Fe(III)heme–HSA–
myristate complex was incubated for 1 h at room tempera-
ture with continuous stirring [28].
Stock solutions of 1.2 · 10
)1
m anti-HIV drugs were pre-
pared by dissolving abacavir, atazanavir and nevirapine in
dimethylsulfoxide. Anti-HIV drugs were added to the
Fe(III)heme–HSA 1.0 · 10
)4
m solution to a final concen-
tration of 1.0 · 10
)4

m.
Automatic flexible ligand-docking simulation to HSA
was performed using autodock 3.0 and the graphical user
interface autodocktools [54–56,68]. The structure of
Fe(III)heme–HSA–myristate was downloaded from the Pro-
tein Data Bank (PDB code: 1O9X) [28]. Ribbon representa-
tion of HSA with stick representation of ligands was drawn
with the swiss-pdbviewer [71]. The nevirapine geometry
was energy-minimized starting from the structure of the
drug observed in its complex with the Thr215Tyr mutant
HIV-1 reverse transcriptase (PDB code: 1LWO) [72].
Abacavir and atazanavir structures were calculated using
the Dundee prodrg server [73]. Single bonds were allowed
to rotate freely during the Monte Carlo-simulated anneal-
ing procedure. Analysis of the conformational space was
restricted to a cubic box of 40 A
˚
, edge centred on the coor-
dinates of heme (for FA1 site) or myristate (for FA2, FA6,
and FA7 sites). Monte Carlo-simulated annealing was per-
formed by starting from a temperature of 900 K with a rel-
ative cooling factor of 0.95 ⁄ cycle, in order to reach the
temperature of 5 K in 100 cycles [56–58].
Binding of Fe(III)heme to HSA in the presence of either
drug and ⁄ or myristate was investigated spectrophotometri-
cally using an optical cell with 1.0 cm path length on a
Cary 50 Bio spectrophotometer (Varian Inc., Palo Alto,
CA). In a typical experiment, a small amount of a
1.2 · 10
)2

m Fe(III)heme solution in 1.0 · 10
)1
m NaOH
was diluted in the optical cell in a solvent mixture of 10%
dimethylsulfoxide (this concentration does not affect differ-
ence spectra) in 1.0 · 10
)1
m phosphate buffer pH 7.0 to a
final chromophore concentration of 1.0 · 10
)5
m, in the
presence of anti-HIV drugs (at 4.0 · 10
)5
m concentration,
i.e. similar to concentrations used in therapy) at different
myristate concentrations (0–1.0 · 10
)4
m). This solution
was titrated with HSA by adding small amounts of a
1.0 · 10
)3
m protein solution in 1.0 · 10
)1
m phosphate
buffer pH 7.0 and recording the spectrum after a few min-
utes incubation following each addition. Difference spectra
with respect to Fe(III)heme were taken and binding iso-
therms were analysed by plotting the difference of absor-
bance against the protein concentration. Data were fitted
by using the following equation:

where DA is the difference in the Soret band (411 nm)
absorbance, DA
max
is the difference of absorbance at limit-
ing HSA concentration, K
a
is the association constant for
Fe(III)heme binding to HSA (i.e. K
À 1
d
), [L
t
] is the total
concentration of Fe(III)heme, [P
t
] is the total concentra-
tion of HSA, and N is the number of equivalent binding
sites.
1
H NMRD profiles of 1.0 · 10
)4
m Fe(III)heme–HSA
were recorded on a Stelar Spinmaster-FFC fast-field cycling
relaxometer (Stelar, Mede, PV, Italy) in the absence
and presence of abacavir (1.0 · 10
)4
m), nevirapine
(1.0 · 10
)4
m) and atazanavir (1.0 · 10

)4
m), in the absence
and presence of 1.0 · 10
)4
and 4.0 · 10
)4
m myristate.
NMRD profiles were obtained by measuring water proton
longitudinal relaxation rates (R
1obs
) at magnetic field
strengths in the range from 2.4 · 10
)4
to 2.35 · 10
)1
T
(corresponding to proton Larmor frequencies from 0.01 to
10 MHz). The R
1p
relaxivity values (i.e. paramagnetic con-
tributions to the solvent water longitudinal relaxation rate
referenced to a 1.0 · 10
)3
m concentration of paramagnetic
agent) were determined by subtracting from the observed
relaxation rate (R
1obs
) the blank relaxation rate value
(R
1dia

) measured for the buffer at the experimental temper-
ature.
Acknowledgements
The authors wish to thank Professor Massimo Coletta
and Professor Riccardo Fesce for helpful discussions.
This study was partly supported by grants from the
Italian Ministry of Health (Istituto Nazionale per le
DA ¼
DA
max
Á

ðK
a
Á½L
t
þNÁ½P
t
ÁK
a
þ 1ÞÀ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðK
a
Á½L
t
þNÁ½P
t
ÁK
a

þ 1Þ
2
À 4K
2
a
Á½L
t
ÁNÁ½P
t

q

2K
a
Á½L
t

ð1Þ
G. Fanali et al. Modulation of HSA ligand binding by anti-HIV drugs
FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS 4499
Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’,
Roma, Italy, ‘Ricerca corrente 2006’ to PA). This paper
is dedicated to the memory of Dr Fabrizio Poccia.
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Supplementary material
The following supplementary material is available
online:
Fig. S1. Binding isotherms for abacavir (A) and
nevirapine (B) binding to HSA (FA free), at pH 7.0
and 25°C.
Fig. S2. Binding isotherms for Fe(III)heme binding to

HSA (FA free) and to HSA–myristate complexes in
the absence of drugs (A) and in the presence of
4.0 · 10
–4
M abacavir (B), 4.0 · 10
–3
M nevirapine (C)
and 4.0 · 10
–4
M atazanavir (D), at pH 7.0 and 25°C.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
Modulation of HSA ligand binding by anti-HIV drugs G. Fanali et al.
4502 FEBS Journal 274 (2007) 4491–4502 ª 2007 The Authors Journal compilation ª 2007 FEBS