Modulation of heme and myristate binding to humanserum albumin by anti-HIV drugs An optical and NMR spectroscopic study Gabriella Fanali1, Alessio Bocedi2,3,*, Paolo Ascenzi2,3and Mauro
Trang 1Modulation of heme and myristate binding to human
serum albumin by anti-HIV drugs
An optical and NMR spectroscopic study
Gabriella Fanali1, Alessio Bocedi2,3,*, Paolo Ascenzi2,3and Mauro Fasano1
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)4m, 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: mauro.fasano@uninsubria.it
*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 Kd 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 Kdvalues of 1· 10)6and 2· 10)6m, respectively Therefore,
at concentrations used in therapy (in the 1–5· 10)6m 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.
Trang 2Sudlow’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, Nfi 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 inhiinhi-bitors 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.
Trang 3in 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 Kd 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 DAmax 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)5m, curves cannot be fitted
using Eqn (1): however, at higher myristate
concentra-tions the Kd 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 Kd¼ 7.4· 10)8m, 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 DAmax value changes with the 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 T1 in the new magnetic field Therefore, measurement of magnetization at progres-sive time intervals allows us to obtain the T1value In order to read the magnetization value, it has to be transformed into an electromagnetic signal by a radio-frequency pulse at its Larmor radio-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.
Trang 4experiment 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)4m 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)5M myri-state; open downward triangles, 7.5 · 10)5M myristate; open diamonds, 1.0 · 10)4M 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
1.0 · 10)5 (5.3 ± 0.4) · 10)7 (2.9 ± 0.4) · 10)7 a a
7.5 · 10)5 (1.3 ± 0.2) · 10)6 (2.5 ± 0.4) · 10)7 (2.9 ± 0.3) · 10)7 a
1.0 · 10)4 (1.3 ± 0.1) · 10)6 (5.1 ± 0.8) · 10)7 (1.1 ± 0.2) · 10)7 a
a The binding isotherm deviates significantly from the hyperbolic behaviour, therefore data cannot be analysed in terms of Eqn (1).
Trang 5region 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
Kdby a factor of two and at the same time modulates
the Nfi 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
FA6 ) 28.9 ) 30.7 ) 11.2
FA7 ) 31.6 ) 33.5 10 3
Trang 6binds to HSA at Kd¼ 10)6m (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.
Trang 7contacting 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 Kd 2 · 10)6m
(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 interacstabiliza-tion 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 R1p 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 R1pvalues 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.
Trang 8viral 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)4M myristate; open diamonds: 4.0 · 10)4M myristate Fe(III)heme–HSA concentration was 1.0 · 10)4M R1pvalues were normalized to 1.0 · 10)3M For further details see text.
Trang 9Fe(III)heme–HSA was prepared by adding the
appropri-ate volume of 1.2· 10)2m Fe(III)heme, dissolved in
1.0· 10)1m NaOH, to a 1.0· 10)4m HSA solution in
0.1 m phosphate buffer pH 7.0 In the final HSA solution
Fe(III)heme–HSA was 1.0· 10)4m
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)1Æcm)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)1m The solution was
heated to 100C and stirred to dissolve the FA The
sodium myristate solution was cooled and then mixed with
1.0· 10)4m 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)1manti-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)4m solution to a final
concen-tration of 1.0· 10)4m
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 representarepresenta-tion 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)2m Fe(III)heme solution in 1.0· 10)1m 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)1mphosphate buffer pH 7.0 to a final chromophore concentration of 1.0· 10)5m, in the presence of anti-HIV drugs (at 4.0· 10)5mconcentration, i.e similar to concentrations used in therapy) at different myristate concentrations (0–1.0· 10)4m) This solution was titrated with HSA by adding small amounts of a 1.0· 10)3m protein solution in 1.0· 10)1m 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, DAmaxis the difference of absorbance at limit-ing HSA concentration, Ka is the association constant for Fe(III)heme binding to HSA (i.e K 1
d ), [Lt] is the total concentration of Fe(III)heme, [Pt] is the total concentra-tion of HSA, and N is the number of equivalent binding sites
1
H NMRD profiles of 1.0· 10)4m 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)4m), nevirapine (1.0· 10)4m) and atazanavir (1.0· 10)4m), in the absence and presence of 1.0· 10)4 and 4.0· 10)4m myristate NMRD profiles were obtained by measuring water proton longitudinal relaxation rates (R1obs) at magnetic field strengths in the range from 2.4· 10)4 to 2.35· 10)1T (corresponding to proton Larmor frequencies from 0.01 to
10 MHz) The R1prelaxivity values (i.e paramagnetic con-tributions to the solvent water longitudinal relaxation rate referenced to a 1.0· 10)3mconcentration of paramagnetic agent) were determined by subtracting from the observed relaxation rate (R1obs) the blank relaxation rate value (R1dia) 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
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Trang 10Malattie 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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